Condensed polycyclic conjugated polymers and their use for biological detection

ABSTRACT

Fluorescent water soluble conjugated polymers including polycyclic aromatic comonomers are provided. The conjugated polymers can be linked to an acceptor fluorescent dye. The conjugated polymers find use in conjugates with biological substrates having applications in a variety of applications including methods of analyte detection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/808,211 filed Feb. 20, 2019, the disclosure of which is hereinincorporated by reference.

FIELD

The invention relates in general to fluorescent conjugated polymers,biological conjugates and their methods of analyte detection.

BACKGROUND

Fluorescent probes are valuable reagents for the analysis and separationof molecules and cells and for the detection and quantification of othermaterials. A very small number of fluorescent molecules can be detectedunder optimal circumstances. Barak and Webb visualized fewer than 50fluorescent lipid analogs associated with the LDL reception of cellsusing a SIT camera, J. CELL BIOL., 90, 595-604 (1981). Flow cytometrycan be used to detect fewer than 10,000 fluorescein molecules associatedwith particles or certain cells (Muirhead, Horan and Poste,BIOTECHNOLOGY, 3, 337-356 (1985)). Some specific examples of theapplication of fluorescent probes are (1) identification and separationof subpopulations of cells in a mixture of cells by the techniques offluorescence flow cytometry, fluorescence-activated cell sorting andfluorescence microscopy; (2) determination of the concentration of asubstance that binds to a second species (e.g., antigen-antibodyreactions) in the technique of fluorescence immunoassay; (3)localization of substances in gels and other insoluble supports by thetechniques of fluorescence staining.

When employing fluorescent polymers for the above purposes, there aremany constraints on the choice of the fluorescent polymer. Oneconstraint is the absorption and emission characteristics of thefluorescent polymer, since many ligands, receptors, and materials in thesample under test, e.g. blood, urine, cerebrospinal fluid, willfluoresce and interfere with an accurate determination of thefluorescence of the fluorescent label. This phenomenon is calledautofluorescence or background fluorescence. Another consideration isthe ability to conjugate the fluorescent polymer to ligands andreceptors and other biological and non-biological materials and theeffect of such conjugation on the fluorescent polymer. In manysituations, conjugation to another molecule may result in a substantialchange in the fluorescent characteristics of the fluorescent polymerand, in some cases, substantially destroy or reduce the quantumefficiency of the fluorescent polymer. It is also possible thatconjugation with the fluorescent polymer will inactivate the function ofthe molecule that is labeled. A third consideration is the quantumefficiency of the fluorescent polymers which should be high forsensitive detection. A fourth consideration is the light absorbingcapability, or extinction coefficient, of the fluorescent polymers,which should also be as large as possible. Also of concern is whetherthe fluorescent molecules will interact with each other when in closeproximity, resulting in self-quenching. An additional concern is whetherthere is non-specific binding of the fluorescent polymers to othercompounds or container walls, either by themselves or in conjunctionwith the compound to which the fluorescent polymer is conjugated.

The applicability and value of the methods indicated above are closelytied to the availability of suitable fluorescent compounds. In recentyears, the rapid advances in lasers and LED technology have provided avariety of laser and LED excitation sources installed in newfluorescence instruments. In particular, there is a need for fluorescentsubstances that have strong absorption and emit fluorescence with alarge Stokes shift, since excitation of these fluorophores produces lessautofluorescence and also multiple chromophores fluorescing at differentwavelengths can be analyzed simultaneously if the full visible and nearinfrared regions of the spectrum can be utilized.

Phycobiliproteins have made an important contribution because of theirhigh extinction coefficient and high fluorescence quantum yield. Thesefluorophore-containing proteins can be covalently linked to manyproteins and are used in fluorescence antibody assays in microscopy andflow cytometry. However, the phycobiliproteins have a few disadvantagesthat limit their biological applications, e.g., (1) thephycobiliproteins are relatively complex and tend to dissociate inhighly diluted solutions; (2) They are extremely unstable and fadequickly upon illumination; (3) the phycobiliproteins have very weakabsorption at 355 nm and 405 nm. Brightly fluorescent conjugatedpolymers permit detection or location of the attached materials withgreat sensitivity. Certain polyconjugated polymers have demonstratedutility as labeling reagents for immunological applications, e.g. U.S.Pat. Nos. 8,110,673; 8,835,113; 9,085,799 to Bazan et al; U.S. Pat. Nos.9,719,998; 9,758,625; 9,547,008; 9,139,869; 8,158,444; 8,455,613;8,354,239; 8,362,193; and 8,575,303 to Gaylord, et al.; U.S. Pat. No.9,896,538 to Diwu et al.; WO2019023463 to Xu et al; WO 2013/101902 toChiu et al. The other biological applications of polyconjugated polymershave been well documented by Thomas III et al. (Chem. Rev. 2007, 107,1339); Zhu et al (Chem. Rev. 2012, 112, 4687) and Zhu et al. (Chem. Soc.Rev., 2011, 40, 3509). However, there are still greatly unmet needs forbright fluorescent probes with strong absorption at the important laserand LED lines with large Stokes shift for the diverse and complicatedbiological applications.

SUMMARY

Fluorescent water soluble conjugated polymers including polycyclicaromatic comonomers are provided. The conjugated polymers can be linkedto an acceptor fluorescent dye. The conjugated polymers find use inconjugates with biological substrates having applications in a varietyof applications including methods of analyte detection.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the presentembodiments will be more fully understood from the following detaileddescription of illustrative embodiments taken in conjunction with theaccompanying drawings in which:

FIG. 1. The normalized absorption spectra of CPCP 11 in PBS buffer(pH=7.4).

FIG. 2. The normalized fluorescence spectra of CPCP 11 in PBS buffer(pH=7.4).

FIG. 3. The normalized absorption spectra of iFluor acceptor dyes (FD)linked to the conjugated polymers in PBS buffer (pH=7.4). The spectradisplayed from left to right are iFluor 350, iFluor 405, iFluor 488,iFluor 514, iFluor 532, iFluor 546, iFluor 568, iFluor 594, iFluor 633,iFluor 647, iFluor 680, iFluor 700, iFluor 750, iFluor 790, iFluor 800,iFluor 810, iFluor 820 and iFluor 860.

FIG. 4. The normalized fluorescence spectra of iFluor acceptor dyes (FD)linked to the conjugated polymers in PBS buffer (pH=7.4). The spectradisplayed from left to right are iFluor 350, iFluor 405, iFluor 488,iFluor 514, iFluor 532, iFluor 546, iFluor 568, iFluor 594, iFluor 633,iFluor 647, iFluor 680, iFluor 700, iFluor 750, iFluor 790, iFluor 800,iFluor 810, iFluor 820 and iFluor 860.

FIG. 5. Jurkat cells are centrifuged to remove medium, and blocked withthe solution of HH buffer containing 1% BSA, 100 ug/ml Goat IgG for 30min. The cells are stained with 0.4 ug/ml mouse IgG (A, control) orCPCP55 (B) for 30-60 min. The cells are washed once to remove stainingsolution, and analyzed with flow cytometer (Pacific Blue channel). (A).Control: mouse IgG (0.4 ug/ml); B. CPCP55 (0.4 ug/ml).

FIG. 6. The cellular F-actin staining with CPCP 60. Hela cells are fixedwith 3-4% formaldehyde in PBS at room temperature for 10-30 minutes. Thefixed cells are washed with PBS buffer for 3 times. 0.1% Triton is addedto the fixed cells to increase conjugate permeability for 10 minutes.The cells are rinsed for 3 times with PBS. 5 μl of CPCP 60 solution(1-100 μg/ml) is added into the fixed cells (100 μL/well, 96-wellplate). The cells are incubated at room temperature for 20 to 90minutes, and rinsed gently with PBS for 3 times to remove excessphalloidin conjugate before imaging under a fluorescence microscope.

FIG. 7A-7B. The fluorescence imaging of tubulin in Hela cells with CPCP51. The Hela cells are fixed with 4% formaldehyde. Mouse anti-tubulin isincubated with the fixed cells. Following the primary incubation, cellsare rinsed with 5 volumes of staining buffer and spun down for 3-5minutes. The cells are then incubated with CPCP 51 at concentrationswithin the range 10 ng/mL-100 ug/mL for 30-60 minutes. Following thesecondary incubation, cells are rinsed with 3-5 volumes of stainingbuffer. The cells are imaged with an Keyence fluorescence microscope.(FIG. 7A). Control without mouse anti-tubulin added; (FIG. 7B). Withmouse anti-tubulin added.

FIG. 8. The normalized absorption spectra of CPCP 140 in PBS buffer(pH=7.4).

FIG. 9. The normalized fluorescence spectra of CPCP 140 in PBS buffer(pH=7.4).

FIG. 10. The Human peripheral blood lymphocytes were stained with CPCP140-labeled anti-Human CD4 (done SK3, mouse IgG1, κ). The fluorescencesignal is monitored using NovoCyte flow cytometer using Pacific BlueChannel. The CPCP 140-labeled anti-Human CD4 conjugate (CPCP 165) isprepared from CPCP 140 succinimidyl ester (CPCP 150) as described inExample 43.

DEFINITIONS

Before the present invention is described in further detail, it is to beunderstood that this invention is not limited to the particularmolecules, methodologies, devices, solutions or apparatuses described,as such methods, devices, solutions or apparatuses can, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention. The following definitionsare set forth to illustrate and define the meaning and scope of thevarious terms used to describe the invention herein.

Use of the singular forms “a” “an” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “a probe” includes a plurality of probes, and the like.Additionally, use of specific plural references, such as “two” “three”etc., read on larger numbers of the same subject unless the contextclearly dictates otherwise.

Terms such as “connected” “attached” “conjugated” and “linked” are usedinterchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise; in one example, the phrase “conjugatedpolymer” is used in accordance with its ordinary meaning in the art andrefers to a polymer containing an extended series of unsaturated bonds,and that context dictates that the term “conjugated” should beinterpreted as something more than simply a direct or indirectconnection, attachment or linkage.

The term “alkyl” as used herein, by itself or as part of another group,refers to straight, branched chain or cyclic radicals having up to 50carbons, unless the chain length or ring size is limited thereto, suchas methyl, ethyl, propyl, cyclopropanyl, isopropyl, butyl, t-butyl,isobutyl, pentyl, hexyl, cyclohexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, and decyl,among others. In some embodiments, an alkyl group includes from 1 to 20carbon atoms. In some embodiments, an alkyl group includes from 1 to 10carbon atoms. In certain embodiments, an alkyl group includes from 1 to6 carbon atoms, such as from 1 to 4 carbon atoms.

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

The term “alkylene” as employed herein, by itself or as part of anothergroup, refers to straight, branched chain or cyclic divalent radicalshaving up to 50 carbons, unless the chain length or ring size is limitedthereto. Typical examples include methylene (—CH₂—), ethylene(—CH₂CH₂—), propylene, butylene, pentylene, hexylene, heptylene,octylene, nonylene, and decylene, among others.

The term “alkenyl” as used herein, by itself or as part of anothergroup, means a straight, branched chain or cyclic radical having 2-50carbon atoms and one or more carbon-carbon double bonds, unless thechain length or ring size is limited thereto, such as ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl,among others. The alkenyl chain may be 2 to 10 carbon atoms in length.Alternatively, the alkenyl chain may be 2 to 4 carbon atoms in length.

The term “alkenylene” as used herein, by itself or as part of anothergroup, means straight, branched chain or cyclic divalent radical having2-50 carbon atoms, unless the chain length or ring size is limitedthereto, said straight, branched chain or cyclic radical containing atleast one carbon-carbon double bond. Typical examples include ethenylene(—CH═CH—), propenylene (—CH═CHCH₂— and —CH₂CH═CH—), n-butenylene, and3-methyl-2-pentenylene, hexenylene, heptenylene, octenylene, nonenylene,and decenylene, among others.

The term “alkynyl” as used herein, by itself or as part of anothergroup, means a straight, branched chain or cyclic radical of 2-50 carbonatoms, unless the chain length or ring size is limited thereto, havingat least one carbon-carbon triple bond between two of the carbon atomsin the chain, such as acetylenyl, 1-propynyl, and 2-propynyl, amongothers. The alkynyl chain may be 2 to 10 carbon atoms in length.Alternatively, the alkynyl chain may be from 2 to 4 carbon atoms inlength.

The term “alkynylene” as used herein, by itself or as part of anothergroup, means a straight, branched chain or cyclic divalent radicalhaving 2-50 carbon atoms, unless the chain length or ring size islimited thereto, that contains at least one carbon-carbon triple bond.Typical examples include ethynylene (—C≡C—), propynylene (—C≡CCH₂— and—CH₂C≡C—), n-butynylene, 4-methyl-2-pentynylene, 1-butynylene,2-butynylene, 3-butynylene, 4-butynylene, pentynylene, hexynylene,heptynylene, octynylene, nonynylene, and decynylene, among others.

The term “alkoxy” as used herein, by itself or as part of another group,refers to any of the above radicals linked via an oxygen atom. Typicalexamples include methoxy, ethoxy, isopropyloxy, sec-butyloxy,n-butyloxy, t-butyloxy, n-pentyloxy, 2-methylbutyloxy, 3-methylbutyloxy,n-hexyloxy, and 2-ethylbutyloxy, among others. Alkoxy also may includePEG groups (—OCH₂CH₂O—) or alkyl moieties that contain more than oneoxygen atom.

The term “aryl” as employed herein, by itself or as part of anothergroup, refers to an aryl or aromatic ring system containing 1 to 10unsaturated rings (each ring containing 6 conjugated carbon atoms and noheteroatoms) that are optionally fused to each other or bonded to eachother by carbon-carbon single bonds, that is optionally furthersubstituted as described below. Examples of aryl ring systems include,but are not limited to, substituted or unsubstituted derivatives offluorenyl, phenyl, biphenyl, o-, m-, or p-terphenyl, 1-naphthyl,2-naphthyl, 1-, 2-, or 9-anthryl, 1-, 2-, 3-, 4-, or 9-phenanthrenyl,1-perylenyl, 1-ovalenyl, 1-benzoperyenyl, 1- or 2-chrysenyl, 1- or2-hexahelicenyl, 1-corannulenyl, 1-coronenyl, 1-, 2- or 4-pyrenyl. Arylgroups of interest include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like. In certain embodiments, an aryl groupincludes from 6 to 20 carbon atoms. In certain embodiments, an arylgroup includes from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

The term “heteroaryl” as employed herein, by itself or as part ofanother group, refers to groups having 5 to 14 ring atoms; 6, 10 or 14 πelectrons shared in a cyclic array; and containing carbon atoms and 1,2, 3, or 4 oxygen, nitrogen or sulfur heteroatoms (where examples ofheteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl,thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl,xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl,3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl,pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl,furazanyl, phenoxazinyl, and tetrazolyl groups.

Any aryl or heteroaryl ring system is unsubstituted or optionally andindependently substituted by any synthetically accessible and chemicallystable combination of substituents, such as H, halogen, cyano, sulfo,alkali or ammonium salt of sulfo, nitro, carboxy, alkyl, perfluoroalkyl,alkoxy, alkylthio, amino, monoalkylamino, dialkylamino or alkylamido,the alkyl portions of which having 18 or fewer carbons.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Substituents of interest include, but are not limited to, alkylenedioxy(such as methylenedioxy), -M, —R⁶⁰, —O⁻, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O—, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹,—C(O)O⁻, —C(S)OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹,—NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹,R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certainembodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O)₂O—, —OS(O)₂R⁶⁰, —P(O)(O)₂, —P(O)(OR⁶⁰)(O), —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹.In certain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻. Incertain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(O)OR⁶⁰, —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above. Whenthe group being substituted is an aryl or heteroaryl group, thesubstituent(s) (e.g., as described herein) may be referred to as “arylsubstituent(s)”.

The term “fluorescent dye or FD” as employed herein, by itself or aspart of another group, refers to an aromatic or heteroaromatic moietythat emits fluorescence longer than 500 nm. The examples of fluorescentdyes include, but not limited to coumarins, fluoresceins, rhodamines,cyanines, bodipys, phthalocyanines, porphyrins, acridines, acridones,DDAO, carbazines, anthrancences, anthraquinones, DRAQ-5, azulenes,benzoxazinones, azacoumarins, benzoimidazoles, benzoxazoles,benzothiazoles, tetrapyrroles, diketopyrrolopyrrole, pyrazolines,hypericins, hypocrellins, perylenequinones, IR-140, luciferin,naphthamides, naphthalenes, naphthoquinones, NBD, SBD, oxazines,oxazoles, dapoxyls, osmium complexes, ruthenium complexes, platinumcomplexes, polycyclic dyes, pyrilium salts, nanocrystals, rhodoles,Schiff bases, squraines, styryls, polythiophenes, tetrazolium salts orupconversion oxides.

The terms “halogen” or “halo” as employed herein, by itself or as partof another group, refers to chlorine, bromine, fluorine or iodine.

The terms “amino” or “amine” include NH₂, “monoalkylamine” or“monoalkylamino” and “dialkylamine” or “dialkylamino”. The terms“monoalkylamine” and “monoalkylamino” “dialkylamine” and “dialkylaminoas employed herein, by itself or as part of another group, refers to thegroup NH₂ where one hydrogen has been replaced by an alkyl group, asdefined above.

The terms “dialkylamine” and “dialkylamino” as employed herein, byitself or as part of another group, refers to the group NH₂ where bothhydrogens have been replaced by alkyl groups, as defined above.

The term “hydroxyalkyl” as employed herein, by itself or as part ofanother group, refers to an alkyl group where one or more hydrogensthereof are substituted by one or more hydroxyl moieties.

The term “haloalkyl” as employed herein, by itself or as part of anothergroup, refers to an alkyl group where one or more hydrogens thereof aresubstituted by one or more halo moieties. Typical examples includechloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl,trichloroethyl, trifluoroethyl, fluoropropyl, and bromobutyl, amongothers.

The term “haloalkenyl” as employed herein, by itself or as part ofanother group, refers to an alkenyl group where one or more hydrogensthereof are substituted by one or more halo moieties.

The term “haloalkynyl” as employed herein, by itself or as part ofanother group, refers to an alkynyl group where one or more hydrogensthereof are substituted by one or more halo moieties.

The term “carboxyalkyl” as employed herein, by itself or as part ofanother group, refers to an alkyl group where one or more hydrogensthereof are substituted by one or more carboxylic acid moieties.

The term “heteroatom” as used herein, by itself or as part of anothergroup, means an oxygen atom (“O”), a sulfur atom (“S”) or a nitrogenatom (“N”). It will be recognized that when the heteroatom is nitrogen,it may form an NR₁R₂ moiety, where R₁ and R₂ are, independently from oneanother, hydrogen or alkyl, or together with the nitrogen to which theyare bound, form a saturated or unsaturated 5-, 6-, or 7-membered ring.

The term “carboxy” as used herein, by itself or as part of anothergroup, is represented by —COOW wherein W is a hydrogen, an alkali metalion, an ammonium or other biologically compatible counter ion.

The term “sulfonate” as used herein, by itself or as part of anothergroup, is represented by —S(═O)₂OW wherein W is a hydrogen, an alkalimetal ion, an ammonium or other biologically compatible counter ion.

The term “phosphonate” as used herein, by itself or as part of anothergroup, is represented by —P(═O)O₂W₂ wherein W is a hydrogen, an alkalimetal ion, an ammonium or other biologically compatible counter ion.

The term “boronate” as used herein, by itself or as part of anothergroup, is represented by —B(OW)₂ wherein W is a hydrogen, an alkalimetal ion, an ammonium or other biologically compatible counter ion.

The term “ammonium” as used herein, by itself or as part of anothergroup, is represented —N(R₃)X wherein n is 1-20, R is a short alkyl(e.g. C₁-C₁₂ alkyl); X is a biologically compatible anion such as F⁻,Cl⁻, Br⁻ or I⁻. Ammonium may include a nitrogen ring structure such aspyridinium, acridinium or quinlonium etc.

The term “sulfonium” as used herein, by itself or as part of anothergroup, is represented —S(R₂)X wherein n is 1-20, R is a short alkyl(e.g. C₁-C₁₂ alkyl);

X is a biologically compatible anion such as F⁻, Cl⁻, Br⁻ or I⁻.

The term “phosphonium” as used herein, by itself or as part of anothergroup, is represented —P(R₃)X wherein n is 1-20, R is a short alkyl(e.g. C₁-C₁₂ alkyl);

X is a biologically compatible anion such as F⁻, Cl⁻, Br⁻ or I⁻.

The term “polyethyleneglycol or PEG” as used herein, by itself or aspart of another group, refers to a polymeric group including a chaindescribed by the formula —(CH₂CH₂O—)_(n)— or a derivative thereof, where“n” is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less,such as 2 to 30, 3 to 15, or 10 to 15. In some cases, n is 2-30. It isunderstood that the PEG polymeric group may be of any convenient lengthand may include a variety of terminal groups and/or further substituentgroups, including but not limited to, alkyl (e.g., methyl), aryl,hydroxyl, amino, acyl, acyloxy, and amido terminal and/or substituentgroups.

The term “water soluble group”, “water solubilizing group” or “WSG” asused herein, by itself or as part of another group, refers to the moietycapable of increasing the water solubility of a polymer. The term WSGrefers to a group that is well solvated in aqueous environments e.g.,under physiological conditions, and that imparts improved watersolubility upon the molecules to which it is attached. WSG include, butare not limited to PEG groups, carboxy, sulfonate, phosphonate,boronate, amine, ammonium, sulfonium, phosphonium, alcohol, or sugar. Ingeneral, one or more WSGs are attached as substituent or sidechaingroups of comonomers of the conjugated polymer.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention. These and other embodiments aredisclosed or are obvious from and encompassed by, the following DetailedDescription.

The term “functional group or FG” as used herein, by itself or as partof another group, is a reactive moiety that can be used to covalentlylink polymers of the invention to a biological target. They include, butnot limited to activated esters, acrylamides, acyl azides, acyl halides,acyl nitriles, aldehydes, ketones, alkyl halides. alkyl sulfonates,anhydrides, aryl halides, aziridines, boronates, carbodiimides,diazoalkanes, epoxides, haloacetamides, haloplatinate, halotriazines,imido esters, isocyanates, isothiocyanates, maleimides,phosphoramidites, silyl halides, sulfonate esters, sulfonyl halides,azides, 1,2,4,5-tetrazines, hydroxylamines, hydrazines, cysteines,azides, nitrile-N-oxides, anthracenes, amines, anilines, thiols,alcohols, phenols, carboxylic acids, glycols, heterocycles, alkynes,cyclooctynes, methyl 2-diphenylphosphinobenzonate, cycloalkynes, orDBCO. A FG can sometimes be referred to as a reactive FG orchemoselective functional group. Any convenient compatible functionalgroups can be used to conjugate molecules of interest to a conjugatedpolymer of this disclosure.

The term “end groups” as used herein, by itself or as part of anothergroup, is the two moieties located on the two terminals of a polymer.They include, but not limited to hydrogen, bromo, iodo, boronyl or a FG.

The term “linker or L” as used herein, by itself or as part of anothergroup, refers to a linking moiety that connects two groups and has abackbone of 100 atoms or less in length. A linker can be a spacer thatlinks the polymer of this invention to a reactive moiety or a biologicaltarget. A linker may be a covalent bond or a chain of between 1 and 100atoms in length, for example a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14,16, 18, 20 or more carbon atoms in length, where the linker may belinear, branched, cyclic or a single atom. In some cases, the linker isa branching linker that refers to a linking moiety that connects threeor more groups. In certain cases, one, two, three, four or five or morecarbon atoms of a linker backbone may be optionally substituted with asulfur, nitrogen or oxygen heteroatom. The bonds between backbone atomsmay be saturated or unsaturated, and in some cases not more than one,two, or three unsaturated bonds are present in a linker backbone. Thelinker may include one or more substituent groups, for example with analkyl, aryl or alkenyl group. A linker may include, without limitations,polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, whichmay be straight or branched, e.g., methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), nbutyl, n-pentyl, 1,1-dimethylethyl(t-butyl), and the like. The linker backbone may include a cyclic group,for example, an aryl, a heterocycle or a cycloalkyl group, where 2 ormore atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included inthe backbone. A linker may be cleavable or non-cleavable. They include,but not limited to alkyl, alkylaryl, alkylheteroaryl, aryl, heteroarylor a PEG. The term “conjugated polymer” as used herein, by itself or aspart of another compound, is a polymer containing an extended series ofrandomly interconnected unsaturated bonds, aryls and/or heteroaryls.

The term “biological substrate or BS” as used herein, by itself or aspart of another group, is a biological target molecule or biomolecule.They include, but not limited to antibodies, antigens, proteins,peptides, oligonucleotides, DNA, RNA, PNA, aptamers, sugars,antibiotics, metabolites, cAMP, cGMP, polysaccharides, viruses, cellsand tissues.

The term “sample” refers to a material or mixture of materials, in somecases in liquid form, containing one or more analytes of interest. Theterm can refer to any plant, animal or bacterial material containingcells or producing cellular metabolites, such as, for example, tissue orfluid isolated from an individual (including without limitation plasma,serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) orfrom in vitro cell culture constituents, as well as samples from theenvironment. The term “sample” may also refer to a “biological sample”.

The terms “determining,” “measuring,” and “assessing,” and “assaying”are used interchangeably and include both quantitative and qualitativedeterminations.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention. These and other embodiments aredisclosed or are obvious from and encompassed by, the following DetailedDescription.

DETAILED DESCRIPTION

The present invention discloses a new type of fluorescent conjugatedpolymer that finds use in preparing biological conjugates, and that hasdesirable properties, such as: (1) high fluorescence quantum yield; (2)red-shifted emission; (3) high water solubility; (4) high linearity; (5)high planarity; (6) high fluorescence resonance energy transfer (FRET)efficiency when a second dye coupled to the polymer; and (7) highphotostability.

The present disclosure provides a water-soluble conjugated polymercontaining a conjugated segment having the structure of Formula 1:

wherein the polymer comprises x different comonomer units (Mr-1 to Mr-x)that are distributed (e.g., randomly) along the polymer main chain orbackbone; wherein Mr-1 is a condensed polycyclic aromatic orheteroaromatic comonomer containing four or more 5-membered and/or6-membered rings (e.g., as described herein); wherein Mr-2 to Mr-x aredifferent and distinct comonomers that are independently a double bond,a triple bond, an aryl, or a heteroaryl; m1 is an integer larger than 5,m2 to mx are integers from 0 to 200, provided that

(1). the sum of m1 to mx is

10; and

(2). at least one of Mr1 to Mr-X has a WSG; and

(3). optionally at least one of Mr1 to Mr-X has a FG or a L-BS.

It is understood the conjugated polymers described herein can includeend groups that cap the terminal co-monomer units. The polymers of thisinvention may be capped on the two terminals by H, a phenyl, asubstituted phenyl, an aryl, a substituted aryl, a substitutedheteroaryl, or a heteroaryl. The end group can be optionally substitutedby bromo, iodo, boronyl, a -L-FG or a L-BS. They are preferably cappedwith a phenyl, substituted phenyl, a fluorene or a substituted analog.

The polycyclic aromatic comonomers of the conjugated polymers of thisdisclosure contain four or more condensed or interconnected 5-memberedand/or 6-membered rings. These polycyclic aromatic ring systems can haveat least 3 or 4 aromatic rings condensed or interconnected (e.g., fused)with a configuration that provides a conjugated system. In some cases,two or three 6 membered aryl or heteroaryl rings are linked viaintervening fused 5-membered carbocyclic or heterocyclic rings. Thepolycyclic aromatic ring system includes a conjugated backbone suitablefor incorporation into a conjugated polymer.

The conjugated polymers of this disclosure include polycyclic aromaticcomonomers that provide structures capable of harvesting light withparticular absorption maximum wavelengths and converting it to emittedlight at longer emission maximum wavelengths. The conjugated polymersare fluorescent. Conjugated polymers (CPs) are characterized by adelocalized electronic structure where the backbone contains conjugatedcomonomer units. The conjugated polymers can be efficient lightharvesting molecules and provide for optical amplification via Forsterresonance energy transfer (FRET) to an acceptor fluorescent dye in closeproximity. Such energy transfer mechanisms from donor conjugated polymerto acceptor dye are relatively short range. The polycyclic aromaticcomonomers can provide conjugated polymers with spectral properties thathave absorption maximum peak longer than 260 nm, and emission maximumpeak longer than 300 nm. These polycyclic aromatic monomers can havefluorescence quantum yield larger than 10%. Some of them are selectivelylisted in Table 1 as examples without limiting the scope of thisinvention.

TABLE 1 Exemplary Mr-1 co-monomers that can be utilized in theconjugated polymers of this disclosure. Each R substituent represents asidechain group which can be incorporated into the comonomer to impartdesirable properties on the co-monomer and resulting conjugatedpolymers, e.g., increased water solubility, or conjugation site, e.g.,to a fluorescent dye or biomolecule. In some embodiments, each R isindependently selected from H, alkyl, substituted alkyl, linker, WSG,-L-WSG, -L-FG, and -L-BS. It is understood that any of the comonomerstructures described in Table 1 may further include one or more arylsubstituents in addition to the depicted R substituent side groups:

The conjugated polymers include x distinct types of co-monomer unitsarranged along the polymer backbone, where multiple units of eachdistinct co-monomer type may be present. In some cases, x is 10 or less,such as 9, 8, 7, 6, 5, 4, 3, or 2. In certain embodiments, x is 1 suchthat the conjugated polymer includes only Mr-1 comonomers. It isunderstood that depending on the method of preparation, a variety ofarrangements of co-monomers are possible in the conjugated polymer,e.g., random or coblock configurations. In some embodiments, thecomonomers are arranged randomly along the backbone. In such cases, theresulting conjugated polymers can be represented by a formula showingthe numbers or mol % of each comonomer unit.

In some embodiments, the conjugated polymer is composed of 5 mol % orgreater of Mr-1 comonomers, such as 10 mol % or greater, 15 mol % orgreater, 20 mol % or greater, 25 mol % or greater, or even more.

In some embodiments of formula 1, Mr-1 is of Formula 2a or Formula 2b

wherein:

the Mr-1 comonomer units are connected to adjacent comonomers of theconjugated polymer backbone through any two positions of C₂, C₃, C₆ andC₇ of the Mr-1 comonomer;

R₁ to R₈ are independently selected from hydrogen, halogen, an alkyl, aWSG, an aryl, a heteroaryl group, a FD, a FG, and L-BS;

A and B are independently selected from O, S, N—R₁₁, P—R₁₁, O═P—R₁₁,O═P—OR₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, O═S—R₁₁, and O═S(O)—R₁₁, wherein R₁₁and R₁₂ are independently selected from hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, and L-BS; and

Y and Z are independently selected from none, C, O, S, N, N—R₁₃, P,P—R₁₃, O═P—R₁₃, O═P—OR₁₃, O═S—R₁₃, and O═S(O)—R₁₃ wherein R₁₃ and R₁₄independently represent hydrogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS;

with the proviso that at least one of A, B, Y and Z is a heteroatom.

In some embodiments of formula 2a, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C2 and C7 of the Mr-1 comonomer. It is understood thatin Formula 2a-11a, at the numbered positions 1-3 and 6-7, an Rsubstituent (i.e., R₁, R₂, R₃, R₆ or R₇) may be present at thosepositions which are no conjugated to an adjacent comonomer or terminalend group. For example, in some embodiments of Formula 2a and 2b, Mr-1has one of the structures Formula 3a and 3b:

where * indicates point of attachment to adjacent co-monomers or aterminal end group and R₃ and R₆ are as defined above. In someembodiments of formula 3a, the Mr-1 comonomer units are connected toadjacent comonomers of the conjugated polymer backbone through positionsC3 and C6 of the Mr-1 comonomer, and formula 3a includes groups R₂ andR₆ are positions C2 and C6.

In some embodiments of formula 1, Mr-1 is of Formula 4a or Formula 4b:

wherein:

the Mr-1 comonomer units are connected to adjacent comonomers of theconjugated polymer backbone through position C2/C3 and position C6/C7 ofthe Mr-1 comonomer;

R₁ to R₈ are independently selected from hydrogen, halogen, an alkyl, aWSG, an aryl, a heteroaryl group, a FD, a FG, and L-BS;

R₁₁ to R₁₄ are independently selected from hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, and L-BS; and

Mr-2 to Mr-x are independently, optionally substituted with a FG orL-BS;

wherein at least one of R₁₁ to R₁₄ is a WSG.

In some embodiments of formula 4a and 4b, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C2 and C6 or C7 of the Mr-1 comonomer:

In some embodiments of formula 4a and 4b, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C3 and C6 or C7 of the Mr-1 comonomer.

In some embodiments of formula 1, Mr-1 is of Formula 6a or Formula 6b:

wherein:

the Mr-1 comonomer units are connected to adjacent monomers of theconjugated polymer backbone through position C2/C3 and position C6/C7 ofthe Mr-1 comonomer;

R₁ to R₈ are independently selected from hydrogen, a halogen, an alkyl,a WSG, an aryl, a heteroaryl group, a FD, a FG, and L-BS;

R₁₁ to R₁₃ are independently selected from hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, and L-BS; Mr-2 to Mr-x areindependently, optionally substituted by a FG or L-BS;

wherein at least one of R₁₁ to R₁₃ is a WSG.

In some embodiments of formula 6a and 6b, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C2 and C7 of the Mr-1 comonomer. In some embodimentsof formula 6a and 6b, the Mr-1 comonomer units are connected to adjacentcomonomers of the conjugated polymer backbone through positions C2 andC6 of the Mr-1 comonomer. In some embodiments of formula 6a and 6b, theMr-1 comonomer units are connected to adjacent comonomers of theconjugated polymer backbone through positions C3 and C6 of the Mr-1comonomer. In some embodiments of formula 6a and 6b, the Mr-1 comonomerunits are connected to adjacent comonomers of the conjugated polymerbackbone through positions C3 and C7 of the Mr-1 comonomer.

In some embodiments of formula 1, Mr-1 is of Formula 7a:

wherein:

the Mr-1 comonomer units are connected to adjacent monomers of theconjugated polymer backbone through position C2/C3 and position C6/C7 ofthe Mr-1 comonomer;

R₁ to R₈ are independently selected from hydrogen, a halogen, an alkyl,a WSG, an aryl, a heteroaryl group, a FD, a FG, and L-BS;

R₁₁ to R₁₃ are independently selected from hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, and L-BS; and Mr-2 to Mr-x areindependently, optionally substituted with a FG, or a L-BS;

wherein at least one of R₁₁ to R₁₃ is a WSG.

In some embodiments of formula 7a, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C2 and C7 of the Mr-1 comonomer. In some embodimentsof formula 7a, the Mr-1 comonomer units are connected to adjacentcomonomers of the conjugated polymer backbone through positions C3 andC6 of the Mr-1 comonomer.

In some embodiments of formula 1, Mr-1 is of Formula 8a:

wherein:

the Mr-1 comonomer units are connected to adjacent monomers of theconjugated polymer backbone through position C2/C3 and position C6/C7 ofthe Mr-1 comonomer;

R₁ to R₈ are independently selected from hydrogen, a halogen, an alkyl,a WSG, an aryl, a heteroaryl group, a FD, a FG, and L-BS;

R₁₁ to R₁₃ are independently selected from hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, and L-BS; and

Mr-2 to Mr-x are independently, optionally substituted by a FG, or aL-BS;

wherein at least one of R₁₁ to R₁₃ is a WSG.

In some embodiments of formula 8a, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C2 and C7 of the Mr-1 comonomer. In some embodimentsof formula 8a, the Mr-1 comonomer units are connected to adjacentcomonomers of the conjugated polymer backbone through positions C3 andC6 of the Mr-1 comonomer.

In some embodiments of formula 1, Mr-1 is of Formula 9a:

wherein:

the Mr-1 comonomer units are connected to adjacent monomers of theconjugated polymer backbone through position C2/C3 and position C6/C7 ofthe Mr-1 comonomer;

R₁ to R₈ are independently selected from hydrogen, a halogen, an alkyl,a WSG, an aryl, a heteroaryl group, a FD, a FG, and L-BS;

R₁₁ to R₁₃ are independently selected from hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, and L-BS; and Mr-2 to Mr-x areindependently, optionally substituted with a FG, or a L-BS;

wherein at least one of R₁₁ to R₁₃ is a WSG.

In some embodiments of formula 9a, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C2 and C7 of the Mr-1 comonomer. In some embodimentsof formula 9a, the Mr-1 comonomer units are connected to adjacentcomonomers of the conjugated polymer backbone through positions C3 andC6 of the Mr-1 comonomer.

In some embodiments of formula 1, Mr-1 is of Formula 10a:

wherein:

the Mr-1 comonomer units are connected to adjacent monomers of theconjugated polymer backbone through position C1/C2 and C6/C7 of the Mr-1comonomer;

R₂ to R₆ are independently selected from hydrogen, a halogen, an alkyl,a WSG, an aryl, a heteroaryl group, a FD, a FG, and L-BS;

A′, B′ and C′ are independently selected from O, S, N—R₁₁, P—R₁₁,O═P—R₁₁, O═P—OR₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, O═S—R₁₁, and O═S(O)—R₁₁wherein R₁₁ and R₁₂ are independently selected from hydrogen, an alkyl,a WSG, an aryl, a heteroaryl group, a FD, a FG, and L-BS;

Y and Z are independently selected from O, S, N—R₁₃, P—R₁₃, O═P—R₁₃,O═P—OR₁₃, O═S—R₁₃, and O═S(O)—R₁₃ wherein R₁₃ is hydrogen, an alkyl, aWSG, an aryl, a heteroaryl group, a FD, a FG, or L-BS;

Mr-2 to Mr-x are independently, optionally substituted with a FG, orL-BS wherein at least one of A′, B′ and C′ comprises a WSG.

In some embodiments of formula 10a, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C1 and C7 of the Mr-1 comonomer. In some embodimentsof formula 10a, the Mr-1 comonomer units are connected to adjacentcomonomers of the conjugated polymer backbone through positions C2 andC6 of the Mr-1 comonomer.

In some embodiments of formula 1, Mr-1 is of Formula 11a:

wherein: the Mr-1 comonomer units are connected to adjacent monomers ofthe conjugated polymer backbone through positions C1/C2 and C7/C6 of theMr-1 comonomer; and at least two of R₁₁ to R₁₆ is a WSG.

In some embodiments of formula 11a, the Mr-1 comonomer units areconnected to adjacent comonomers of the conjugated polymer backbonethrough positions C1 and C7 of the Mr-1 comonomer. In some embodimentsof formula 11a, the Mr-1 comonomer units are connected to adjacentcomonomers of the conjugated polymer backbone through positions C2 andC6 of the Mr-1 comonomer.

The Mr-2 to Mr-x comonomers of the invention typically contains at leastone double bond or one triple bond that provides for pi-conjugation toadjacent co-monomers in the conjugated polymer backbone. In someembodiments, one of the comonomers is ethenylene (—CH═CH—) orsubstituted ethenylene. In some embodiments, one of the comonomers isacetylene (—CC—). In some embodiments, the co-monomer is an optionallysubstituted aryl or heteroaryl co-monomer. The Mr-2 to Mr-x comonomersmay be selected to impart a desirable absorption wavelength upon theresulting conjugated polymer. Exemplary comonomer groups include, butare not limited to, substituted or unsubstituted derivatives of phenyl,biphenyl, o-, m-, or p-terphenyl, 1-naphthyl, 2-naphthyl, 1-, 2-, or9-anthryl, 1-, 2-, 3-, 4-, or 9-phenanthrenyl and 1-, 2- or 4-pyrenyl,perylenyl, thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl,thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl,xanthenyl, phenoxathinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl,3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl,pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl,furazanyl, phenoxazinyl, and tetrazolyl. In some embodiments, anoptionally substituted aryl or heteroaryl co-monomer is selected fromsubstituted or unsubstituted 1,4-phenyl, a substituted or unsubstituted1,3-phenyl, a substituted or unsubstituted 4,4′-biphenyl, a substitutedor unsubstituted 2,5-pyridyl, and a substituted or unsubstituted2,6-pyridyl.

The conjugated polymer may have any convenient length. In some cases,the total number of monomeric repeating units of the conjugated polymermay fall within the range of 5 to 10,000, 10 to 10,000, 100 to 10,000units or segments. In some embodiments, the total number of monomericrepeating units of the conjugated polymer may fall within the range of 5to 1,000, such as 5 to 500, 5 to 400, 5 to 300, 5 to 200, or 5 to 100units. The length of the conjugated polymer can be determined by thetotal numbers (mx) of each type of co-monomer (Mr-x) present in thepolymer backbone. In some embodiments of formula 1, the sum of m1 to mxis 10 or more, such as 20 or more, 30 or more, 40 or more, 50 or more,100 or more, up to 10,000 or less, such as up to 5000, up to 2000 or upto 1000.

The subject conjugated polymer may be water soluble, or comprise aplurality of WSGs. Any convenient water solubilizing groups (WSGs) maybe included in the to provide for increased water-solubility. While theincrease in solubility may vary, in some cases the increase is 2 fold ormore, e.g., 5 fold, 10 fold, 25 fold, 50 fold, 100 fold or more ascompared to the polymer without the WSGs. The WSGs may be charged, e.g.,positively or negatively charged. In certain cases, the WSG is a neutralhydrophilic group. In some embodiments, the WSG is a hydrophilicpolymer, e.g., a polyethylene glycol, a cellulose, a chitosan, or aderivative thereof. The WSG may be attached via a linker. WSGs ofinterest include, but are not limited to, carboxylate, phosphonate,phosphate, sulfonate, sulfate, sulfinate, sulfonium, ester, polyethyleneglycols (PEG) and modified PEGs, hydroxyl, amine, ammonium, guanidinium,pyridinium, polyamine and sulfonium, polyalcohols, straight chain orcyclic saccharides, primary, secondary, tertiary, or quaternary aminesand polyamines, phosphonate groups, phosphinate groups, ascorbategroups, glycols, including, polyethers, —COOM′, —SO₃M′, —PO₃M′, —NR₃ ⁺,Y′, (CH₂CH₂O)_(p)R and mixtures thereof, where Y′ can be any halogen,sulfate, sulfonate, or oxygen containing anion, p can be 1 to 500, eachR can be independently H or an alkyl (such as methyl) and M′ can be acationic counterion or hydrogen, —(CH₂CH₂O)_(yy)CH₂CH₂XR^(yy),—(CH₂CH₂O)_(yy)CH₂CH₂X—, —X(CH₂CH₂O)_(yy)CH₂CH₂—, glycol, andpolyethylene glycol, wherein yy is selected from 1 to 1000, X isselected from O, S, and NR^(xx), and R^(zz) and R^(yy) are independentlyselected from H and C₁₋₃ alkyl.

Multiple WSGs may be included at a single location via a branchinglinker. In some embodiments, the conjugated polymer includessubstitutent(s) selected from an alkyl, an aralkyl and a heterocyclicgroup, each group further substituted with a include water solubilizinggroups hydrophilic polymer group, such as a polyethylglycol (PEG) (e.g.,a PEG of 2-20 units).

In some embodiments, the conjugated polymer itself is used as afluorescent molecule for detecting target analytes. In some embodiments,the conjugated polymer is linked to a fluorescent dye, e.g., aconventional small molecule dye, such that FRET can occur from the donorpolymer to the acceptor dye. The dye can be linked or configured inenergy receiving proximity to the conjugated polymer. The acceptorfluorescent dye can be selected to provide for a desirable emissionwavelength from the resulting donor polymer-acceptor dye conjugate.

The fluorescent dyes (FDs) linked to the conjugated polymers of theinvention is typically a FD that has absorption maximum longer than 400nm, and emission maximum longer than 450 nm with fluorescence quantumyield larger than 10%. They are typically selected from coumarins,styryls, fluoresceins, rhodamines, cyanines, BODIPYs or other polycyclicaromatics. Many of them are commercially available as selectively listedin Table 2 as examples.

TABLE 2 Exemplary acceptor fluorescent dyes (FDs) that can be linked tothe conjugated polymers Absorption Emission Fluorophore (nm) (nm) ATTO465 453 508 ATTO 488 501 523 ATTO 495 495 527 ATTO 514 511 533 ATTO 532532 553 ATTO 550 554 576 ATTO 565 563 592 ATTO 590 594 624 ATTO 594 601627 ATTO 610 615 634 ATTO 620 619 643 ATTO 633 629 657 ATTO 647 645 669ATTO 647N 644 669 ATTO 655 663 684 ATTO 665 663 684 ATTO 680 680 700ATTO 700 700 719 ATTO 725 729 752 ATTO 740 740 7645-carboxy-2,7-dichlorofluorescein 504 529 5-Carboxyfluorescein (5-FAM)492 518 5-Carboxynapthofluorescein 598 668 5-Carboxytetramethylrhodamine(5-TAMRA) 542 568 5-FAM (5-Carboxyfluorescein) 492 518 5-ROX 578 6046-TAMRA 548 568 CF 555 555 565 CF 568 562 583 CF 594ST 593 614 CF 633630 650 CF 640R 642 662 CF 647 650 665 CF 660C 667 685 CF 680 681 698CF680R 680 701 CF 750 755 777 CF 770 770 797 CF 790 784 806 CL-NERF 504540 CMFDA 494 520 Cy2 489 506 Cy3 554 568 Cy3.5 581 598 Cy5 649 666Cy5.5 675 695 Cy7 743 767 DDAO 646 659 DiA 456 591 DiD 644 665 DiI 549565 DyLight 488 493 518 DyLight 550 562 576 DyLight 594 593 618 DyLight633 638 568 DyLight 650 652 672 DyLight 680 692 712 DyLight 755 754 776DyLight 800 777 794 DiO 487 502 DiR 748 780 DM-NERF 497 540 DsRed 558583 DTAF 494 520 DY-490 491 515 DY-495 494 521 DY-505 507 528 DY-530 533554 DY-547 558 573 DY-548 558 572 DY-549 562 577 DY-549P1 563 578 DY-550562 577 DY-554 544 570 DY-555 547 573 DY-556 548 574 DY-560 560 578DY-590 581 600 DY-591 581 598 DY-594 594 615 DY-605 600 624 DY-610 610632 DY-615 623 643 DY-630 638 658 DY-631 637 657 DY-632 636 658 DY-633638 658 DY-634 636 657 DY-635 648 670 DY-636 647 670 DY-647 653 673DY-648 655 676 DY-649 656 670 DY-649P1 654 672 DY-650 656 676 DY-651 655677 DY-652 653 676 DY-654 653 677 DY-675 675 699 DY-676 675 699 DY-677674 698 DY-678 674 694 DY-679 679 698 DY-679P1 679 697 DY-680 691 709DY-681 692 709 DY-682 692 709 DY-700 707 728 DY-701 709 730 DY-703 705721 DY-704 706 721 DY-730 734 755 DY-731 736 755 DY-732 735 756 DY-734733 755 DY-749 759 780 DY-750 751 774 DY-751 752 772 DY-752 750 771DY-754 748 771 DY-776 772 787 DY-777 770 788 DY-778 767 787 DY-780 783799 DY-781 784 796 DY-782 785 794 DY-800 777 791 DY-831 844 875 Eosin524 545 Erythrosin 529 555 FITC 490 520 fluo-3 506 520 fluo-4 494 516Fluor-Ruby 555 582 FluorX 494 520 FM 1-43 479 598 FM 4-46 515 640 iFluor488 498 520 iFluor 555 558 578 iFluor 594 588 610 iFluor 647 649 670iFluor 680 686 702 iFluor 700 696 720 iFluor 750 755 785 iFluor 780 787808 Lyso Tracker Green 504 511 Lyso Tracker Yellow 551 576 MitotrackerGreen 490 516 Mitotracker Orange 551 576 Mitotracker Red 578 516 NBD 466539 Oregon Green 488 494 517 Oregon Green 514 506 526 PKH26 551 567PKH67 496 520 Resorufin 571 584 RH 414 532 716 Rhod-2 552 576 Rhodamine550 573 Rhodamine 110 496 520 Rhodamine 123 507 529 Rhodamine 6G 525 555Rhodamine B 540 625 Rhodamine Green 502 527 Rhodamine Red 570 590 RoseBengal 525 550 Spectrum Green 497 538 Spectrum Orange 559 588 SpectrumRed 587 612 SYTO 11 508 527 SYTO 12 499 522 SYTO 13 488 509 SYTO 14 517549 SYTO 15 516 546 SYTO 16 488 518 SYTO 17 621 634 SYTO 18 490 507 SYTO20 512 530 SYTO 21 494 517 SYTO 22 515 535 SYTO 23 499 520 SYTO 24 490515 SYTO 25 521 556 SYTO 40 420 441 SYTO 41 430 454 SYTO 42 433 460 SYTO43 436 467 SYTO 44 446 471 SYTO 45 452 484 SYTO 59 622 645 SYTO 60 652678 SYTO 61 628 645 SYTO 62 652 676 SYTO 63 657 673 SYTO 64 599 619 SYTO80 531 545 SYTO 81 530 544 SYTO 82 541 560 SYTO 83 543 559 SYTO 84 567582 SYTO 85 567 583 SYTOX Blue 445 470 SYTOX Green 504 523 SYTOX Orange547 570 Texas Red 595 620 Tide Fluor 2 (TF2) 500 527 Tide Fluor 2WS(TF2WS) 502 525 Tide Fluor 3 (TF3) 555 584 Tide Fluor 3WS (TF3WS) 555565 Tide Fluor 4 (TF4) 590 618 Tide Fluor 5WS (TF5WS) 649 664 Tide Fluor6WS (TF6WS) 676 695 Tide Fluor 7WS (TF7WS) 749 775 Tide Fluor 8WS(TF8WS) 775 807 TRITC 550 573 XTRITC 582 601

Many embodiments of the compounds of the invention possess an overallelectronic charge. It is to be understood that when such electroniccharges are shown to be present, they are balanced by the presence ofappropriate counterions, which may or may not be explicitly identified.A biologically compatible counterion, which is preferred for someapplications, is not toxic in biological applications, and does not havea substantially deleterious effect on biomolecules. Where the compoundof the invention is positively charged, the counterion is typicallyselected from, but not limited to, chloride, bromide, iodide, sulfate,alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylboride, nitrate and anions of aromatic oraliphatic carboxylic acids. Where the compound of the invention isnegatively charged, the counterion is typically selected from, but notlimited to, alkali metal ions, alkaline earth metal ions, transitionmetal ions, ammonium or substituted ammonium or pyridinium ions.Preferably, any necessary counterion is biologically compatible, is nottoxic as used, and does not have a substantially deleterious effect onbiomolecules. Counterions are readily changed by methods well known inthe art, such as ion-exchange chromatography, or selectiveprecipitation.

It is to be understood that the polymer conjugates of the invention havebeen drawn in one or another particular electronic resonance structure.Every aspect of the instant invention applies equally to polymerconjugates that are formally drawn with other permitted resonancestructures, as the electronic charge on the subject polymer conjugatesis delocalized throughout the polymer conjugate itself.

Also provided are conjugates between the subject conjugated polymers anda biological substrate (BS). The BS can be linked to the conjugatedpolymer by any convenient means at a variety of sites, such as viaconjugation to a terminal linker (-L-FG) or via conjugation to asidechain group of a comonomer (-L-FG). In another preferred embodimentof the invention, the polymer conjugate contains at least one L-BS orL-FD-BS, where BS is attached to the polymer by a well-known reaction aslisted in Table 3 as examples. In certain embodiments, the covalentlinkage attaching the polymer to BS contains multiple intervening atomsthat serve as a Linker (L). The polymers can be used to label a widevariety of biological, organic or inorganic substances that contain orare modified to contain functional groups with suitable reactivity,resulting in chemical attachment of the conjugated substances.

TABLE 3 Examples of compatible reactive functional groups (e.g.,chemoselective functional groups) for preparing the biologicalconjugates of the subject polymers. Functional Group Matching FunctionalResulting covalent (FG) Group linkages activated esters amines/anilinescarboxamides acrylamides thiols thioethers acyl azides amines/anilinescarboxamides acyl halides amines/anilines carboxamides acyl halidesalcohols/phenols esters acyl nitriles alcohols/phenols esters acylnitriles amines/anilines carboxamides aldehydes amines/anilines iminesaldehydes or ketones hydrazines hydrazones aldehydes or ketoneshydroxylamines oximes alkyl halides amines/anilines alkyl amines alkylhalides carboxylic acids esters alkyl halides thiols thioethers alkylhalides alcohols/phenols ethers alkyl sulfonates thiols thioethers alkylsulfonates carboxylic acids esters alkyl sulfonates alcohols/phenolsethers anhydrides alcohols/phenols esters anhydrides amines/anilinescarboxamides aryl halides thiols thioethers aryl halides amines arylamines aziridines thiols thioethers boronates glycols boronate esterscarbodiimides carboxylic acids N-acylureas or anhydrides diazoalkanescarboxylic acids esters epoxides thiols thioethers haloacetamides thiolsthioethers haloplatinate amino platinum complex haloplatinateheterocycle platinum complex haloplatinate thiol platinum complexhalotriazines amines/anilines aminotriazines halotriazinesalcohols/phenols triazinyl ethers imido esters amines/anilines amidinesisocyanates amines/anilines ureas isocyanates alcohols/phenols urethanesisothiocyanates amines/anilines thioureas maleimides thiols thioethersphosphoramidites alcohols phosphite esters silyl halides alcohols silylethers sulfonate esters amines/anilines alkyl amines sulfonate estersthiols thioethers sulfonate esters carboxylic acids esters sulfonateesters alcohols ethers sulfonyl halides amines/anilines sulfonamidessulfonyl halides phenols/alcohols sulfonate esters azides alkynes1,2,3-triazoles 1,2,4,5-tetrazines cyclooctynes pyradazineshydroxylamines aldehydes/ketones oxamines hydrazines aldehydes/ketoneshydrazones cysteines aldehydes/ketones thiazolidines aryl azides methyl2-diphenyl- 2-diphenylphosphonyl- phosphinobenzonate benzoamidesNitrile-N-oxides cycloalkynes isoxazoles anthracenes maleimidessuccinimides

Choice of the linkage used to attach the polymer to a biologicalsubstrate to be conjugated typically depends on the functional group onthe biological substrate to be conjugated and the type or length ofcovalent linkage desired. The types of functional groups typicallypresent on the organic or inorganic biological substrates include, butare not limited to, amines, amides, thiols, alcohols, phenols,aldehydes, ketones, phosphonates, imidazoles, hydrazines,hydroxylamines, disubstituted amines, halides, epoxides, carboxylateesters, sulfonate esters, purines, pyrimidines, carboxylic acids,olefinic bonds, azide, alkyne, tetrazine or a combination of thesegroups. A single type of reactive site may be available on thebiological substrate (typical for polysaccharides), or a variety ofsites may occur (e.g. amines, thiols, alcohols, phenols), as is typicalfor proteins. A conjugated biological substrate may be conjugated tomore than one polymer conjugate, which may be the same or different, orto a biological substrate that is additionally modified by a hapten,such as biotin. Alternatively multiple substrates might be conjugated toa single polymer. Although some selectivity can be obtained by carefulcontrol of the reaction conditions, selectivity of labeling is bestobtained by selection of an appropriate reactive polymer conjugate.

Typically, a conjugated polymer including a suitable reactive functionalgroup will react with an amine, a thiol, an alcohol, an aldehyde or aketone. Preferably, the conjugated polymer reactive functional groupreacts with an amine, a thiol functional or a clickable group. In oneembodiment, polymer includes, or is modified to include an acrylamide, areactive amine (including a cadaverine or ethylenediamine), an activatedester of a carboxylic acid (typically a succinimidyl ester of acarboxylic acid), an acyl azide, an acyl nitrile, an aldehyde, an alkylhalide, an anhydride, an aniline, an aryl halide, an azide, anaziridine, a boronate, a carboxylic acid, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide,tetrazine, azide, alkyne or a thiol group. By “reactive platinumcomplex” is particularly meant chemically reactive platinum complexessuch as described in U.S. Pat. Nos. 5,580,990; 5,714,327 and 5,985,566.

Where the conjugated polymer includes a photoactivatable functionalgroup, such as an azide, diazirinyl, azidoaryl, or psoralen derivative,the polymer becomes chemically reactive only after illumination withlight of an appropriate wavelength. Where the conjugated polymerincludes an activated ester of a carboxylic acid, the reactive polymeris particularly useful for preparing polymer conjugates of proteins,nucleotides, oligonucleotides, or haptens. Where the conjugated polymerincludes a maleimide or haloacetamide the reactive polymer isparticularly useful for conjugation to thiol-containing biologicalsubstrates. Where the conjugated polymer includes a hydrazide, thereactive polymer is particularly useful for conjugation toperiodate-oxidized carbohydrates and glycoproteins, and in addition isan aldehyde-fixable polar tracer for cell microinjection. Where theconjugated polymer includes a clickable functional group, the reactivepolymer is particularly useful for conjugation to the complimentaryclickable substrate. Preferably, the conjugated polymer includes acarboxylic acid, a succinimidyl ester of a carboxylic acid, ahaloacetamide, a hydrazine, an isothiocyanate, a maleimide group, analiphatic amine, a perfluorobenzamido, an azidoperfluorobenzamido group,or a psoralen. More preferably, polymer is a succinimidyl ester of acarboxylic acid, a maleimide, an iodoacetamide, or a reactive platinumcomplex.

Based on the above-mentioned attributes, the appropriate reactivepolymers of the invention are selected for the preparation of thedesired polymer conjugates (e.g., FD conjugate and/or BS conjugates ofthe subject conjugated polymers), whose advantageous properties makethem useful for a wide variety of applications. Particularly usefulpolymer conjugates include, among others, conjugates where substrate isa peptide, a nucleotide, an antigen, a steroid, a vitamin, a drug, ahapten, a metabolite, a toxin, an environmental pollutant, an aminoacid, a protein, a nucleic acid, a nucleic acid polymer, a carbohydrate,a lipid, an ion-complexing moiety, a glass or a non-biological polymer.Alternatively, substrate is a cell, a cellular system, a cellularfragment, or a subcellular particle (e.g. inter alia), a virus particle,a bacterial particle, a virus component, a biological cell (such asanimal cell, plant cell, bacteria, yeast, or protist), or a cellularcomponent. Reactive polymers typically label functional groups at thecell surface, in cell membranes, organelles, or cytoplasm.

Typically, the substrate (e.g., BS) is an amino acid, a peptide, aprotein, a tyramine, a polysaccharide, an ion-complexing moiety, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten,a psoralen, a drug, a hormone, a lipid, a lipid assembly, a polymer, apolymeric microparticle, a biological cell or virus. More typically,substrate is a peptide, a protein, a nucleotide, an oligonucleotide, ora nucleic acid. When conjugating polymer conjugates of the invention tosuch biopolymers, it is possible to incorporate more polymers permolecule to increase the fluorescent signal. For polymer-antibodyconjugates, one polymer/antibody is preferred.

In one embodiment, the substrate is an amino acid (including those thatare protected or are substituted by phosphonates, carbohydrates, or C₁to C₂₅ carboxylic acids), or is a polymer of amino acids such as apeptide or protein. Preferred conjugates of peptides contain at leastfive amino acids, more preferably 5 to 36 amino acids. Preferredpeptides include, but are not limited to, neuropeptides, cytokines,toxins, protease substrates, and protein kinase substrates. Preferredprotein conjugates include enzymes, antibodies, lectins, glycoproteins,histones, albumins, lipoproteins, avidin, streptavidin, protein A,protein G, phycobiliproteins and other fluorescent proteins, hormones,toxins, chemokines and growth factors. In one preferred aspect, theconjugated protein is a polymer antibody conjugate.

In one aspect of the invention, the substrate is a conjugated biologicalsubstrate that is an antibody (including intact antibodies, antibodyfragments, and antibody sera, etc.), an amino acid, an angiostatin orendostatin, an avidin or streptavidin, a biotin (e.g. an amidobiotin, abiocytin, a desthiobiotin, etc.), a blood component protein (e.g. analbumin, a fibrinogen, a plasminogen, etc.), a dextran, an enzyme, anenzyme inhibitor, an IgG-binding protein (e.g. a protein A, protein G,protein A/G, etc.), a fluorescent protein (e.g. a phycobiliprotein, anaequorin, a green fluorescent protein, etc.), a growth factor, ahormone, a lectin (e.g. a wheat germ agglutinin, a conconavalin A,etc.), a lipopolysaccharide, a metal-binding protein (e.g. a calmodulin,etc.), a microorganism or portion thereof (e.g. a bacteria, a virus, ayeast, etc.), a neuropeptide and other biologically active factors (e.g.a dermorphin, a deltropin, an endomorphin, an endorphin, a tumornecrosis factor etc.), a non-biological microparticle (e.g. offerrofluid, gold, polystyrene, etc.), a nucleotide, an oligonucleotide,a peptide toxin (e.g. an apamin, a bungarotoxin, a phalloidin, etc.), aphospholipid-binding protein (e.g. an annexin, etc.), a small-moleculedrug (e.g. a methotrexate, etc.), a structural protein (e.g. an actin, afibronectin, a laminin, a microtubule-associated protein, a tublin,etc.), or a tyramide.

In another preferred embodiment, substrate is a nucleic acid base,nucleoside, nucleotide or a nucleic acid polymer, including those thatare modified to possess an additional linker or spacer for attachment ofthe polymer conjugates of the invention, such as an alkynyl linkage(U.S. Pat. No. 5,047,519), an aminoallyl linkage (U.S. Pat. No.4,711,955), or a heteroatom-substituted linker (U.S. Pat. No. 5,684,142)or other linkage. In another preferred embodiment, the conjugatedbiological substrate is a nucleoside or nucleotide analog that links apurine or pyrimidine base to a phosphate or polyphosphate moiety througha noncyclic spacer. In another preferred embodiment, the polymerconjugate is conjugated to the carbohydrate portion of a nucleotide ornucleoside, typically through a hydroxyl group but additionally througha thiol or an amino group (U.S. Pat. Nos. 5,659,025; 5,668,268;5,679,785). Typically, the conjugated nucleotide is a nucleosidetriphosphate or a deoxynucleoside triphosphate or a dideoxynucleosidetriphosphate. Incorporation of methylene moieties or nitrogen or sulfurheteroatoms into the phosphate or polyphosphate moiety is also useful.Nonpurine and nonpyrimidine bases such as 7-deazapurines (U.S. Pat. No.6,150,510) and nucleic acids containing such bases can also be coupledto polymer conjugates of the invention. Nucleic acid adducts prepared byreaction of depurinated nucleic acids with amine, hydrazide orhydroxylamine derivatives provide an additional means of labeling anddetecting nucleic acids, e.g. “A method for detecting abasic sites inliving cells: age-dependent changes in base excision repair.” Atamna H,Cheung I, Ames B N. PROC. NATL. ACAD. SCI. U.S.A. 97, 686-691 (2000).

Preferred nucleic acid polymer conjugates are labeled, single- ormulti-stranded, natural or synthetic DNA or RNA, DNA or RNAoligonucleotides, or DNA/RNA hybrids, or incorporate an unusual linkersuch as morpholine derivatized phosphates, or peptide nucleic acids suchas N-(2-aminoethyl)glycine units. When the nucleic acid is a syntheticoligonucleotide, it typically contains fewer than 50 nucleotides, moretypically fewer than 25 nucleotides. Conjugates of peptide nucleic acids(PNA) (Nielsen, et al. U.S. Pat. No. 5,539,082) may be preferred forsome applications because of their generally faster hybridization rates.

In one embodiment, the conjugated oligonucleotides of the invention areaptamers for a particular target molecule, such as a metabolite, polymerconjugate, hapten, or protein. That is, the oligonucleotides have beenselected to bind preferentially to the target molecule. Methods ofpreparing and screening aptamers for a given target molecule have beenpreviously described and are known in the art [for example, U.S. Pat.No. 5,567,588 to Gold (1996)].

In another preferred embodiment, substrate is a carbohydrate that istypically a polysaccharide, such as a dextran, heparin, glycogen,amylopectin, mannan, inulin, starch, agarose and cellulose.Alternatively, the carbohydrate is a polysaccharide that is alipopolysaccharide. Preferred polysaccharide conjugates are dextran, orlipopolysaccharide conjugates.

Conjugates having an ion-complexing moiety serve as indicators forcalcium, sodium, magnesium, zinc, potassium, or other biologicallyimportant metal ions. Preferred ion-complexing moieties are crown ethers(U.S. Pat. No. 5,405,975); derivatives of1,2-bis-(2-aminophenoxyethane)-N,N,N′,N′-tetraacetic acid (BAPTAchelators; U.S. Pat. Nos. 5,453,517; 5,516,911 and 5,049,673);derivatives of 2-carboxymethoxyaniline-N,N-di-acetic acid (APTRAchelators; AM. J. PHYSIOL., 256, C540 (1989)); or pyridine- andphenanthroline-based metal ion chelators (U.S. Pat. No. 5,648,270); orderivatives of nitrilotriacetic acid, see e.g. “Single-step synthesisand characterization of biotinylated nitrilotriacetic acid, a uniquereagent for the detection of histidine-tagged proteins immobilized onnitrocellulose”, McMahan S A and Burgess R R, ANAL. BIOCHEM., 236,101-106 (1996). Preferably, the ion-complexing moiety is a crown etherchelator, a BAPTA chelator, an APTRA chelator or a derivative ofnitrilotriacetic acid.

Other conjugates of non-biological materials include polymerconjugate-conjugates of organic or inorganic polymers, polymeric films,polymeric wafers, polymeric membranes, polymeric particles, or polymericmicroparticles (magnetic and non-magnetic microspheres); iron, gold orsilver particles; conducting and non-conducting metals and non-metals;and glass and plastic surfaces and particles. Conjugates are optionallyprepared by copolymerization of a polymer conjugate that contains anappropriate functionality while preparing the polymer, or by chemicalmodification of a polymer that contains functional groups with suitablechemical reactivity. Other types of reactions that are useful forpreparing polymer conjugate-conjugates of polymers include catalyzedpolymerizations or copolymerizations of alkenes and reactions of dieneswith dienophiles, transesterifications or transaminations. In anotherpreferred embodiment, the conjugated biological substrate is a glass orsilica, which may be formed into an optical fiber or other structure.

In one embodiment, conjugates of biological polymers such as peptides,proteins, oligonucleotides, nucleic acid polymers are also labeled withat least a second fluorescent dye conjugate, which is optionally anadditional polymer conjugate of the present invention, to form anenergy-transfer pair. In some aspects of the invention, the labeledconjugate functions as an enzyme substrate, and enzymatic hydrolysisdisrupts the energy transfer. In another preferred embodiment of theinvention, the energy-transfer pair that incorporates a polymerconjugate of the invention is conjugated to an oligonucleotide thatdisplays efficient fluorescence quenching in its hairpin conformation[the so-called “molecular beacons” of Tyagi, et al., NATUREBIOTECHNOLOGY, 16, 49 (1998)] or fluorescence energy transfer.

The preparation of polymer conjugates using reactive polymer conjugatesis well documented, e.g. e.g. U.S. Pat. Nos. 8,158,444; 8,455,613;8,354,239; 8,362,193; and 8,575,303 to Gaylord, et al.; also WO2013/101902 to Chiu et al. The other biological applications ofpolyconjugated polymers have been well documented by Thomas III et al.(Chem. Rev. 2007, 107, 1339); Zhu et al (Chem. Rev. 2012, 112, 4687) andZhu et al. (Chem. Soc. Rev., 2011, 40, 3509). Conjugates typicallyresult from mixing appropriate reactive polymers and biologicalsubstrate to be conjugated in a suitable solvent in which both aresoluble. The polymer conjugates of the invention are readily soluble inaqueous solutions, facilitating conjugation reactions with mostbiological materials. For those reactive polymer conjugates that arephotoactivated, conjugation requires illumination of the reactionmixture to activate the reactive polymer conjugates.

The present disclosure provides a conjugated polymer containing thestructure of Formula 2:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through the two positions ofC2, C3, C6 or C7 of the first monomer; wherein R₁ to R₈ independentlyrepresent hydrogen, a halogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS; A and B are independently O, S, N—R₁₁,P—R₁₁, O═P—R₁₁, O═P—OR₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, O═S—R₁₁, O═S(O)—R₁₁wherein R₁₁ and R₁₂ independently represent hydrogen, an alkyl, a WSG,an aryl, a heteroaryl group, a FD, a FG, or a L-BS; Y and Z areindependently none, C, O, S, N, N—R₁₃, P, P—R₁₃, O═P—R₁₃, O═P—OR₁₃,O═S—R₁₃, O═S(O)—R₁₃ wherein R₁₃ and R₁₄ independently representhydrogen, an alkyl, a WSG, an aryl, a heteroaryl group, a FD, a FG, or aL-BS; Mr-2 to Mr-x are independently a double bond, a triple bond, anaryl, a heteroaryl, or a FD that is optionally substituted by a FG, or aL-BS; m1 is an integer larger than 5, m2 to mx are integers from 0 to200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(2). at least one of Mr1 to Mr-X has a WSG;

(3). at least one of Mr1 to Mr-X has a FG or a L-BS;

(4). at least one of A, B Y or Z is a heteroatom

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 2, wherein R₁ to R₈ independently represent hydrogenor fluoro; A and B are independently N—R₁₁, P—R₁₁, O═P—R₁₁, O═P—OR₁₁,R₁₁—C—R₁₂, R₁₁—Si—R₁₂, R₁₁—S—R₁₂, O═S—R₁₁, O═S(O)—R₁₁; R₁₁ and R₁₂independently represent hydrogen, an alkyl, a PEG, a FD, a FG, or a L-BSwherein L is an alkyl or a PEG; Y and Z are independently none orcarbon; Mr-2 to Mr-x are independently a double bond, a triple bond, anaryl, a heteroaryl or, a FD that is optionally substituted by a FG, or aL-BS wherein L is an alkyl or a PEG; m1 is an integer larger than 5, m2to mx are integers from 0 to 200, provided that m1 is

10, and the sum of Mr1 to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 2, wherein R₁ to R₈ independently represent hydrogen;A and B are independently N—R₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, R₁₁—S—R₁₂; R₁₁and R₁₂ are independently a hydrogen, an alkyl, a PEG, a fluorescentdye, a FG, or a L-BS wherein linker (L) is an alkyl or a PEG; Y and Zare independently none or carbon wherein m and n are independently 0 or1; m1 to mx are integers from 1 to 200; provided that the sum of Mr1 toMr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 2, wherein R₁ to R₈ independently represent hydrogen;A and B are independently N—R₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, R₁₁—S—R₁₂; R₁₁and R₁₂ are a PEG; Y and Z are independently none or carbon wherein mand n are independently 0 or 1; Mr-2 is a fluorene that is optionallysubstituted with a FD, a FG, or a L-BS wherein L is an alkyl or a PEG;m1 and m2 are integers from 1 to 200; provided that m1 is

30, and the sum of Mr1 to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 2, wherein R₁ to R₈ independently represent hydrogen;A and B are independently N—R₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, R₁₁—S—R₁₂; R₁₁and R₁₂ are a PEG; Y and Z are independently none or carbon wherein mand n are independently 0 or 1; Mr-2 is a phenyl that is optionallysubstituted with a FD, a FG, or a L-BS wherein L is an alkyl or a PEG;m1 and m2 are integers from 1 to 200; provided that m1 is

30, and the sum of Mr1 to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 2, wherein R₁ to R₈ independently represent hydrogen;A and B are independently N—R₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, R₁₁—S—R₁₂; R₁₁and R₁₂ are a PEG; Y and Z are independently none or carbon wherein mand n are independently 0 or 1; Mr-2 is a FD that is optionallysubstituted with a FG, or a L-BS wherein L is an alkyl or a PEG; Mr-3 isa phenyl that is optionally substituted with a FG, or a L-BS wherein Lis an alkyl or a PEG; m1, m2 and m3 are integers from 1 to 200; providedthat m1 is

30, and the sum of Mr1 to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 2, wherein R₁ to R₈ independently represent hydrogen;A and B are independently N—R₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, R₁₁—S—R₁₂; R₁₁and R₁₂ are a PEG; Y and Z are independently none or carbon wherein mand n are independently 0 or 1; Mr-2 is a FD that is optionallysubstituted with a FG, or a L-BS wherein L is an alkyl or a PEG; m1 andm3 are integers from 1 to 200; provided that m1 is

30, and the sum of Mr1 to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymerscontaining Formula 2, wherein PEGs are PEG6 to PEG18.

In another embodiment, the disclosure provides the conjugated polymerscontaining Formula 2, wherein WSGs are a PEG, a carboxyalkyl, asulfonylalkyl, a phosphonylalkyl, a hydroxyalkyl, an aminoalkyl or anammoniumylalkyl.

In another embodiment, the disclosure provides the conjugated polymerscontaining Formula 2, wherein FG is an activated ester, an aldehyde, anmaleimide, a 1,2,4,5-tetrazine, a hydroxylamine, a hydrazine, an azide,an alkyne, a cyclooctyne, or a DBCO.

In another embodiment, the disclosure provides the conjugated polymerscontaining Formula 2, wherein end group is a hydrogen, bromo, iodo,boronyl or a FG.

In another embodiment, the disclosure provides the conjugated polymerscontaining Formula 2, wherein BS is an antibody, an antigen, a protein,a peptide, an oligonucleotide, a DNA, an RNA, a PNA, an aptamer or acell.

In another embodiment, the disclosure provides the conjugated polymerscontaining Formula 2, wherein FD is a bodipy, a polythiophene, apolypyrrole, a porphyrin, a phthalocyanine, or a carbocyanine.

The present disclosure provides a conjugated polymer containing thestructure of Formula 4:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through C2/C3 and C6/C7 ofthe first monomer; wherein R₁ to R₈ independently represent hydrogen, ahalogen, an alkyl, a WSG, an aryl, a heteroaryl group, a FD, a FG, or aL-BS; R₁₁ to R₁₄ independently represent hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, or a L-BS; Mr-2 to Mr-x areindependently a double bond, a triple bond, an aryl, a heteroaryl, or aFD that is optionally substituted by a FG, or a L-BS; m1 is an integerlarger than 5, m2 to mx are integers from 0 to 200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(2). at least one of R₁₁ to R₁₄ is a WSG; and

(3). at least one of Mr1 to Mr-X has a FG or a L-BS.

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 4, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₄independently represent hydrogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS; Mr-2 to Mr-x are independently a doublebond, a triple bond, an aryl, a heteroaryl, or a FD that is optionallysubstituted by a FG, or a L-BS; m1 is an integer larger than 5, m2 to mxare integers from 0 to 200, provided that m1 is the

10, sum of Mr1 to Mr-X is 50 to 200, and at least one of R₁₁ to R₁₄ is aWSG.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 4, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₄independently represent hydrogen, an alkyl, a PEG, a FD, a FG, or aL-BS; Mr-2 is a fluorene that is optionally substituted with a FD, a FG,or a L-BS wherein L is an alkyl or a PEG; m1 and m2 are integers from 1to 200; provided that m1 is

30, the sum of Mr1 to Mr-X are 10-200 and at least one of R₁₁ to R₁₄ isa PEG.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 4, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₄ are aPEG that is optionally substituted with a FD, a FG, or a L-BS; Mr-2 is aphenyl that is optionally substituted with a FD, a FG, or a L-BS; m1 andm2 are integers from 1 to 200; provided that m1 is

30, the sum of Mr1 to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 4, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₄ are aPEG that is optionally substituted with a FD, a FG, or a L-BS; Mr-2 is aFD that is optionally substituted with a FG, or a L-BS wherein L is analkyl or a PEG; Mr-3 is a phenyl that is optionally substituted with aFG, or a L-BS; m1, m2 and m3 are integers from 1 to 200; provided thatm1 is

30, and the sum of Mr1 to Mr-X are 10-200.

The present disclosure provides a conjugated polymer containing thestructure of Formula 5:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through C2 and C7 of thefirst monomer; wherein R₁ to R₈ independently represent hydrogen, ahalogen, an alkyl, a WSG, an aryl, a heteroaryl group, a FD, a FG, or aL-BS; R₁₁ to R₁₄ independently represent hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, or a L-BS; Mr-2 to Mr-x areindependently a double bond, a triple bond, an aryl, a heteroaryl, or aFD that is optionally substituted by a FG, or a L-BS; m1 is an integerlarger than 5, m2 to mx are integers from 0 to 200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(2). at least one of R₁₁ to R₁₄ is a WSG; and

(3). at least one of Mr1 to Mr-X has a FG or a L-BS.

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 5, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₄independently represent hydrogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS; Mr-2 to Mr-x are independently a doublebond, a triple bond, an aryl, a heteroaryl, or a FD that is optionallysubstituted by a FG, or a L-BS; m1 is an integer larger than 5, m2 to mxare integers from 0 to 200, provided that m1 is

10, the sum of Mr1 to Mr-X is 50 to 200, and at least one of R₁₁ to R₁₄is a WSG.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 5, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₄independently represent hydrogen, an alkyl, a PEG, a FD, a FG, or aL-BS; Mr-2 is a fluorene that is optionally substituted with a FD, a FG,or a L-BS wherein L is an alkyl or a PEG; m1 and m2 are integers from 1to 200; provided that m1 is

30, the sum of Mr1 to Mr-X are 10-200 and at least one of R₁₁ to R₁₄ isa PEG.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 5, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₄ are aPEG that is optionally substituted with a FD, a FG, or a L-BS; Mr-2 is aphenyl that is optionally substituted with a FD, a FG, or a L-BS; m1 andm2 are integers from 1 to 200; provided that m1 is

30, the sum of Mr1 to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 5, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₄ are aPEG that is optionally substituted with a FD, a FG, or a L-BS; Mr-2 is aFD that is optionally substituted by a FG, or a L-BS wherein L is analkyl or a PEG; Mr-3 is a phenyl that is optionally substituted with aFG, or a L-BS; m1, m2 and m3 are integers from 1 to 200; provided thatm1 is

30, and the sum of Mr1 to Mr-X are 10-200.

The present disclosure provides a conjugated polymer containing thestructure of Formula 6:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through C2/C3 and C6/C7 ofthe first monomer; wherein R₁ to R₈ independently represent hydrogen, ahalogen, an alkyl, a WSG, an aryl, a heteroaryl group, a FD, a FG, or aL-BS; R₁₁ to R₁₃ independently represent hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, or a L-BS; Mr-2 to Mr-x areindependently a double bond, a triple bond, an aryl, a heteroaryl, or aFD that is optionally substituted by a FG, or a L-BS; m1 is an integerlarger than 5, m2 to mx are integers from 0 to 200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(2). at least one of R₁₁ to R₁₃ is a WSG; and

(3). at least one of Mr1 to Mr-X has a FG or a L-BS.

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 6, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₃independently represent hydrogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS; Mr-2 to Mr-x are independently a doublebond, a triple bond, an aryl, a heteroaryl, or a FD that is optionallysubstituted by a FG, or a L-BS; m1 is an integer larger than 5, m2 to mxare integers from 0 to 200, provided that m1 is ≥10, the sum of Mr1 toMr-X is 50 to 200, and at least one of R₁₁ to R₁₃ is a WSG.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 6, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₃independently represent hydrogen, an alkyl, a PEG, a FD, a FG, or aL-BS; Mr-2 is a fluorene that is optionally substituted with a FD, a FG,or a L-BS wherein L is an alkyl or a PEG; m1 and m2 are integers from 1to 200; provided that m1 is ≥30, the sum of Mr1 to Mr-X are 10-200 andat least one of R₁₁ to R₁₃ is a PEG.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 6, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₃ are aPEG that is optionally substituted with a FD, a FG, or a L-BS; Mr-2 is aphenyl that is optionally substituted with a FD, a FG, or a L-BS; m1 andm2 are integers from 1 to 200; provided that m1 is ≥30, the sum of Mr1to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 6, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₃ are aPEG that is optionally substituted with a FD, a FG, or a L-BS; Mr-2 is aFD that is optionally substituted by a FG, or a L-BS wherein L is analkyl or a PEG; Mr-3 is a phenyl that is optionally substituted with aFG, or a L-BS; m1, m2 and m3 are integers from 1 to 200; provided thatm1 is ≥30, and the sum of Mr1 to Mr-X are 10-200.

The present disclosure provides a conjugated polymer containing thestructure of Formula 7:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through C2/C3 and C6/C7 ofthe first monomer; wherein R₁ to R₈ independently represent hydrogen, ahalogen, an alkyl, a WSG, an aryl, a heteroaryl group, a FD, a FG, or aL-BS; R₁₁ to R₁₃ independently represent hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, or a L-BS; Mr-2 to Mr-x areindependently a double bond, a triple bond, an aryl, a heteroaryl, or aFD that is optionally substituted by a FG, or a L-BS; m1 is an integerlarger than 5, m2 to mx are integers from 0 to 200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(2). at least one of R₁₁ to R₁₃ is a WSG; and

(3). at least one of Mr1 to Mr-X has a FG or a L-BS.

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 7, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₃independently represent hydrogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS; Mr-2 to Mr-x are independently a doublebond, a triple bond, an aryl, a heteroaryl, or a FD that is optionallysubstituted by a FG, or a L-BS; m1 is an integer larger than 5, m2 to mxare integers from 0 to 200, provided that m1 is

10, the sum of Mr1 to Mr-X is 50 to 200, and at least one of R₁₁ to R₁₃is a WSG.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 7, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₃independently represent hydrogen, an alkyl, a PEG, a FD, a FG, or aL-BS; Mr-2 is a fluorene that is optionally substituted with a FD, a FG,or a L-BS wherein L is an alkyl or a PEG; m1 and m2 are integers from 1to 200; provided that m1 is

30, the sum of Mr1 to Mr-X are 10-200 and at least one of R₁₁ to R₁₃ isa PEG.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 7, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₃ are aPEG that is optionally substituted with a FD, a FG, or a L-BS; Mr-2 is aphenyl that is optionally substituted with a FD, a FG, or a L-BS; m1 andm2 are integers from 1 to 200; provided that m1 is

30, the sum of Mr1 to Mr-X are 10-200.

In another embodiment, the disclosure provides the conjugated polymercontaining Formula 7, wherein R₁ to R₈ are hydrogen; R₁₁ to R₁₃ are aPEG that is optionally substituted with a FD, a FG, or a L-BS; Mr-2 is aFD that is optionally substituted by a FG, or a L-BS wherein L is analkyl or a PEG; Mr-3 is a phenyl that is optionally substituted with aFG, or a L-BS; m1, m2 and m3 are integers from 1 to 200; provided thatm1 is

30, and the sum of Mr1 to Mr-X are 10-200.

The present disclosure provides a conjugated polymer containing thestructure of Formula 8:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through C2/C3 and C6/C7 ofthe first monomer; wherein R₁ to R₁₀ independently represent hydrogen, ahalogen, an alkyl, a WSG, an aryl, a heteroaryl group, a FD, a FG, or aL-BS; R₁₁ to R₁₃ independently represent hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, or a L-BS; Mr-2 to Mr-x areindependently a double bond, a triple bond, an aryl, a heteroaryl, or aFD that is optionally substituted by a FG, or a L-BS; m1 is an integerlarger than 5, m2 to mx are integers from 0 to 200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(2). at least one of R₁₁ to R₁₃ is a WSG; and

(3). at least one of Mr1 to Mr-X has a FG or a L-BS.

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

The present disclosure provides a conjugated polymer containing thestructure of Formula 9:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through C2/C3 and C6/C7 ofthe first monomer; wherein R₁ to R₁₀ independently represent hydrogen, ahalogen, an alkyl, a WSG, an aryl, a heteroaryl group, a FD, a FG, or aL-BS; R₁₁ to R₁₃ independently represent hydrogen, an alkyl, a WSG, anaryl, a heteroaryl group, a FD, a FG, or a L-BS; Mr-2 to Mr-x areindependently a double bond, a triple bond, an aryl, a heteroaryl, or aFD that is optionally substituted by a FG, or a L-BS; m1 is an integerlarger than 5, m2 to mx are integers from 0 to 200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(2). at least one of R₁₁ to R₁₃ is a WSG; and

(3). at least one of Mr1 to Mr-X has a FG or a L-BS.

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

The present disclosure provides a conjugated polymer containing thestructure of Formula 10:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through the two positions ofC1/C2 and C6/C7 of the first monomer; wherein R₂ to R₆ independentlyrepresent hydrogen, a halogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS; A, B and C are independently O, S, N—R₁₁,P—R₁₁, O═P—R₁₁, O═P—OR₁₁, R₁₁—C—R₁₂, R₁₁—Si—R₁₂, O═S—R₁₁, O═S(O)—R₁₁wherein R₁₁ and R₁₂ independently represent hydrogen, an alkyl, a WSG,an aryl, a heteroaryl group, a FD, a FG, or a L-BS; Y and Z areindependently O, S, N—R₁₃, P—R₁₃, O═P—R₁₃, O═P—OR₁₃, O═S—R₁₃, O═S(O)—R₁₃wherein R₁₃ represents hydrogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS; Mr-2 to Mr-x are independently a doublebond, a triple bond, an aryl, a heteroaryl, or a FD that is optionallysubstituted by a FG, or a L-BS; m1 is an integer larger than 5, m2 to mxare integers from 0 to 200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(2). at least one of A, B and C has a WSG; and

(3). at least one of Mr1 to Mr-X has a FG or a L-BS.

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

The present disclosure provides a conjugated polymer containing thestructure of Formula 11:

wherein the polymer comprises monomer units that are randomlydistributed along the polymer main chain with the first monomerconnected to other monomers (Mr-2 to Mr-x) through the two positions ofC1 and C7 of the first monomer; wherein R₃ to R₅ independently representhydrogen, a halogen, an alkyl, a WSG, an aryl, a heteroaryl group, a FD,a FG, or a L-BS; R₁₁ to R₁₆ independently represent hydrogen, an alkyl,a WSG, an aryl, a heteroaryl group, a FD, a FG, or a L-BS; Y and Z areindependently O, S, N—R₁₇, P—R₁₇, O═P—R₁₇, O═P—OR₁₇, O═S—R₁₇, O═S(O)—R₁₇wherein R₁₇ represents hydrogen, an alkyl, a WSG, an aryl, a heteroarylgroup, a FD, a FG, or a L-BS; Mr-2 to Mr-x are independently a doublebond, a triple bond, an aryl, a heteroaryl, or a FD that is optionallysubstituted by a FG, or a L-BS; m1 is an integer larger than 5, m2 to mxare integers from 0 to 200, provided that

(1). the sum of Mr1 to Mr-X is

10; and

(3). at least two of R₁₁ to R₁₆ is a WSG; and

(3). at least one of Mr1 to Mr-X has a FG or a L-BS.

The polymers of this invention may be capped on the two terminals by aphenyl, an aryl, or a heteroaryl that is optimally substituted by bromo,iodo, boronyl, a FG or a L-BS. They are preferably capped with a phenyl,or a fluorene or their substituted analogs.

The present disclosure further provides a method of detecting an analytein a sample, comprising

-   -   a) combining said sample with a detection reagent comprising a        polymer conjugate containing the structure of Formula 1, 2, 3,        4, 5, 6, 7, 8, 9, 10 or 11 under conditions under which said        detection reagent will bind said analyte; and    -   b) detecting the detection reagent bound analyte by        fluorescence,

In one embodiment, the disclosure provides the polymer conjugate ofFormula 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 wherein BS is an antibody.

In another embodiment, the disclosure provides the polymer conjugate ofFormula 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 wherein BS is ananti-digoxigenin antibody.

In another embodiment, the disclosure provides the polymer conjugate ofFormula 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 wherein BS is a goatanti-mouse IgG antibody, goat anti-rabbit IgG antibody, goat anti-humanIgG antibody, donkey anti-mouse IgG antibody, donkey anti-rabbit IgGantibody, donkey anti-human IgG antibody, chicken anti-mouse IgGantibody, chicken anti-rabbit IgG antibody, or chicken anti-human IgGantibody.

In one embodiment, the disclosure provides the polymer conjugate ofFormula 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 wherein BS is an avidin,streptavidin, neutravidin, avidin, or avidin.

In another embodiment, the disclosure provides the polymer conjugate ofFormula 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 wherein the analyte is atarget protein expressed on a cell surface.

In another embodiment, the sample is present on or in solid orsemi-solid matrix. In one aspect of the invention, the matrix is amembrane.

In another aspect, the matrix is an electrophoretic gel, such as is usedfor separating and characterizing nucleic acids or proteins, or is ablot prepared by transfer from an electrophoretic gel to a membrane.

In another aspect, the matrix is a silicon chip or glass slide, and theanalyte of interest has been immobilized on the chip or slide in anarray (e.g. the sample comprises proteins or nucleic acid polymers in amicroarray). In yet another aspect, the matrix is a microwell plate ormicrofluidic chip, and the sample is analyzed by automated methods,typically by various methods of high-throughput screening, such as drugscreening.

The polymer conjugates of the invention are generally utilized bycombining a polymer conjugate of the invention as described above withthe sample of interest under conditions selected to yield a detectableoptical response. The term “polymer conjugate” is used herein to referto all aspects of the claimed polymer conjugates. The polymer conjugatetypically forms a covalent association or complex with an element of thesample, or is simply present within the bounds of the sample or portionof the sample. The sample is then illuminated at a wavelength selectedto elicit the optical response. Typically, staining the sample is usedto determine a specified characteristic of the sample by furthercomparing the optical response with a standard or expected response.

A detectable optical response means a change in, or occurrence of, anoptical signal that is detectable either by observation orinstrumentally. Typically the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The degree and/orlocation of staining, compared with a standard or expected response,indicates whether and to what degree the sample possesses a givencharacteristic.

For biological applications, the polymer conjugates of the invention aretypically used in an aqueous, mostly aqueous or aqueous-misciblesolution prepared according to methods generally known in the art. Theexact concentration of polymer conjugate is dependent upon theexperimental conditions and the desired results. The optimalconcentration is determined by systematic variation until satisfactoryresults with minimal background fluorescence are accomplished.

The polymer conjugates are most advantageously used to stain sampleswith biological components. The sample may comprise heterogeneousmixtures of components (including intact cells, cell extracts, bacteria,viruses, organelles, and mixtures thereof), or a single component orhomogeneous group of components (e.g. natural or synthetic amino acids,nucleic acids or carbohydrate polymers, or lipid membrane complexes).These polymer conjugates are generally non-toxic to living cells andother biological components, within the concentrations of use.

The polymer conjugate is combined with the sample in any way thatfacilitates contact between the polymer conjugate and the samplecomponents of interest. Typically, the polymer conjugate or a solutioncontaining the polymer conjugate is simply added to the sample. Certainpolymer conjugates of the invention tend to be impermeant to membranesof biological cells, and once inside viable cells are typically wellretained. Treatments that permeabilize the plasma membrane, such aselectroporation, shock treatments or high extracellular ATP can be usedto introduce selected polymer conjugates into cells. Alternatively,selected polymer conjugates can be physically inserted into cells, e.g.by pressure microinjection, scrape loading, patch clamp methods, orphagocytosis.

Polymer conjugates that incorporate an aliphatic amine or a hydrazineresidue can be microinjected into cells, where they can be fixed inplace by aldehyde fixatives such as formaldehyde or glutaraldehyde. Thisfixability makes such polymer conjugates useful for intracellularapplications such as neuronal tracing.

Polymer conjugates that possess a lipophilic substituent, such asphospholipids, will non-covalently incorporate into lipid assemblies,e.g. for use as probes for membrane structure; or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials; or for tracing. Lipophilic polymer conjugates areuseful as fluorescent probes of membrane structure.

Using polymer conjugates to label active sites on the surface of cells,in cell membranes or in intracellular compartments such as organelles,or in the cell's cytoplasm, permits the determination of their presenceor quantity, accessibility, or their spatial and temporal distributionin the sample. Photoreactive polymer conjugates can be used similarly tophotolabel components of the outer membrane of biological cells or asphoto-fixable polar tracers for cells.

Optionally, the sample is washed after staining to remove residual,excess or unbound polymer conjugate. The sample is optionally combinedwith one or more other solutions in the course of staining, includingwash solutions, permeabilization and/or fixation solutions, andsolutions containing additional detection reagents. An additionaldetection reagent typically produces a detectable response due to thepresence of a specific cell component, intracellular biologicalsubstrate, or cellular condition, according to methods generally knownin the art. Where the additional detection reagent has, or yields aproduct with, spectral properties that differ from those of the subjectpolymer conjugates, multi-color applications are possible. This isparticularly useful where the additional detection reagent is a polymerconjugate or polymer conjugate-conjugate of the present invention havingspectral properties that are detectably distinct from those of thestaining polymer conjugate.

The polymer conjugates of the invention are used according to methodsextensively known in the art; e.g. use of antibody conjugates inmicroscopy and immunofluorescent assays; and nucleotide oroligonucleotide conjugates for nucleic acid hybridization assays andnucleic acid sequencing (e.g., U.S. Pat. No. 5,332,666 to Prober, et al.(1994); U.S. Pat. No. 5,171,534 to Smith, et al. (1992); U.S. Pat. No.4,997,928 to Hobbs (1991); and WO Appl. 94/05688 to Menchen, et al.).Polymer conjugate-conjugates of multiple independent polymer conjugatesof the invention possess utility for multi-color applications.

At any time after or during staining, the sample is illuminated with awavelength of light selected to give a detectable optical response, andobserved with a means for detecting the optical response. Equipment thatis useful for illuminating the polymer conjugates of the inventionincludes, but is not limited to, hand-held ultraviolet lamps, mercuryarc lamps, xenon lamps, lasers and laser diodes. These illuminationsources are optionally integrated into laser scanners, fluorescencemicroplate readers, standard or minifluorometers, or chromatographicdetectors. Preferred embodiments of the invention are polymer conjugatesthat are be excitable at or near the wavelengths 405 nm.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic films, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined using a flow cytometer, examination of the sample optionallyincludes sorting portions of the sample according to their fluorescenceresponse.

One aspect of the instant invention is the formulation of kits thatfacilitate the practice of various assays using any of the polymerconjugates of the invention, as described above. The kits of theinvention typically comprise a fluorescent polymer conjugate of theinvention where the conjugated biological substrate is a specificbinding pair member, or a nucleoside, a nucleotide, an oligonucleotide,a nucleic acid polymer, a peptide, or a protein. The kit optionallyfurther comprises one or more buffering agents, typically present as anaqueous solution. The kits of the invention optionally further compriseadditional detection reagents, a purification medium for purifying theresulting labeled biological substrate, luminescence standards, enzymes,enzyme inhibitors, organic solvent, or instructions for carrying out anassay of the invention.

EXAMPLES

Examples of some synthetic strategies for selected polymer conjugates ofthe invention, as well as their characterization, synthetic precursors,conjugates and methods of use are provided in the examples below.Further modifications and permutations will be obvious to one skilled inthe art. The examples below are given so as to illustrate the practiceof this invention. They are not intended to limit or define the entirescope of this invention. It is to be understood that this invention isnot limited to particular aspects described, as such may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular aspects only, and is not intended to belimiting since the scope of the present invention will be limited onlyby the appended claims. Where a range of values is provided, it isunderstood that each intervening value, to the tenth of the unit of thelower limit unless the context clearly dictates otherwise, between theupper and lower limit of that range and any other stated or interveningvalue in that stated range, is encompassed within the invention. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges and are also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

Synthesis of the reactive polymers of the invention depends on initialpreparation of certain key intermediates as illustrated in FIG. 3. Forsimplicity, all but a few of the possible substituents are shown ashydrogen. These basic structures are optionally further substituted,during or after synthesis, to give the corresponding polymer conjugatesubstituents as defined above. It is recognized that there are manypossible variations that may yield equivalent results.

The methods for the synthesis of polymers that contain a variety ofreactive functional groups such as those described in Table 3 are welldocumented in the art. Particularly useful are amine-reactive polymerconjugates such as “activated esters” of carboxylic acids, which aretypically synthesized by coupling a carboxylic acid to a relativelyacidic “leaving group”. Other preferred amine-reactive groups includesulfonyl halides, which are prepared from sulfonic acids using ahalogenating agent such as PCl₅ or POCl₃; halotriazines, which areprepared by the reaction of cyanuric halides with amines; andisocyanates or isothiocyanates, which are prepared from amines andphosgene or thiophosgene, respectively. Polymers containing azide,alkyne and tetrazine are particularly useful for conjugation to clickgroup-modified substrates such as the antibodies modified by a clickgroup-containing activated esters.

Polymers containing amines and hydrazides are particularly useful forconjugation to carboxylic acids, aldehydes and ketones. Most often theseare synthesized by reaction of an activated ester of a carboxylic acidor a sulfonyl halide with a diamine, such as cadaverine, or with ahydrazine. Alternatively, aromatic amines are commonly synthesized bychemical reduction of a nitroaromatic compound. Amines and hydrazinesare particularly useful precursors for synthesis of thiol-reactivehaloacetamides or maleimides by standard methods.

In one aspect of the invention, the polymer conjugates of the inventionare used to directly stain or label a sample so that the sample can beidentified or quantitated. For instance, such polymer conjugates may beadded as part of an assay for a biological target analyte, as adetectable tracer element in a biological or non-biological fluid; orfor such purposes as photodynamic therapy of tumors, in which a polymerconjugated sample is irradiated to selectively destroy tumor cells andtissues; or to photoablate arterial plaque or cells, usually through thephotosensitized production of singlet oxygen. In one preferredembodiment, polymer conjugate is used to stain a sample that comprises aligand for which the conjugated biological substrate is a complementarymember of a specific binding pair (e.g. Table 4).

Typically, the sample is obtained directly from a liquid source or as awash from a solid material (organic or inorganic) or a growth medium inwhich cells have been introduced for culturing, or a buffer solution inwhich cells have been placed for evaluation. Where the sample comprisescells, the cells are optionally single cells, including microorganisms,or multiple cells associated with other cells in two or threedimensional layers, including multicellular organisms, embryos, tissues,biopsies, filaments, biofilms, etc.

Alternatively, the sample is a solid, optionally a smear or scrape or aretentate removed from a liquid or vapor by filtration. In one aspect ofthe invention, the sample is obtained from a biological fluid, includingseparated or unfiltered biological fluids such as urine, cerebrospinalfluid, blood, lymph fluids, tissue homogenate, interstitial fluid, cellextracts, mucus, saliva, sputum, stool, physiological secretions orother similar fluids. Alternatively, the sample is obtained from anenvironmental source such as soil, water, or air; or from an industrialsource such as taken from a waste stream, a water source, a supply line,or a production lot.

TABLE 4 Representative specific binding pairs Antigen Antibody BiotinAnti-biotin or avidin or streptavidin or neutravidin IgG* Protein A orprotein G or anti-IgG antibody Drug Drug receptor Toxin Toxin receptorCarbohydrate Lectin or carbohydrate receptor Peptide Peptide receptorNucleotide Complimentary nucleotide Protein Protein receptor Enzymesubstrate Enzyme DNA (RNA) aDNA (aRNA)** Hormone Hormone receptorPsoralen Nucleic acid Target molecule RNA or DNA aptamer Ion Ionchelator *IgG is an immunoglobulin; **aDNA and aRNA are the antisense(complementary) strands used for hybridization

Example 1. The Preparation of Compound 2

Compound 1 is prepared as described by Hsiao, Chien-Chi et al, AdvancedSynthesis & Catalysis, 2010, 352(18), 3267-3274. To the solution ofCompound 1 (3.97 g) and TBAB (1.00 g) in toluene (80 mL) is addedt-butyl acrylate (11.4 mL) in ice bath under Ar protection, followed bythe addition of 50% NaOH (80 mL). The mixture is stirred at roomtemperature for 2.5 hours and ice-water is added. The reaction mixtureis extracted by dichloromethane twice, dried with sodium sulfate,filtered and concentrated. The residue is purified by flashchromatography (hexane-chloroform, 0-10%) to give compound 2 as a whitesolid.

Example 2. The Preparation of Compound 3

To the solution of Compound 2 (8.33 g) and anisole (9.4 mL) indichloromethane (80 mL) are added trifluoroacetic acid (40 mL). Thereaction mixture is stirred at room temperature for 5 hours and thenconcentrated, azeotroped with dichloromethane twice. The residue istriturated with ether (100 mL), filtered and dried to give compound 3 asa white solid.

Example 3. The Preparation of Compound 4

Compound 3 (1.03 g) is sonicated in DMF (30 mL). To the DMF solution,bromine (0.2 mL) is added in ice bath and the reaction mixture isstirred at room temperature. More bromine (0.2 mL each, four times) isadded until complete conversion to the desired product. The mixture ispoured into a sodium sulfite solution (7.5 g in 500 mL water) withstirring. The white precipitate is collected, washed with 0.1%trifluoroacetic acid solution and lyophilized to give compound 4 as awhite solid.

Example 4. The Preparation of Compound 5

To the solution of Compound 4 (2.43 g) in tetrahydrofuran (80 mL) isadded Et₃N (5.82 mL), then ethyl chloroformate (2 mL) in ice bath. Thereaction mixture is stirred at room temperature for 1 hour and filtered.The filtrate is concentrated and dissolved in tetrahydrofuran (90 mL).To the tetrahydrofuran solution NaBH₄ (1.05 g) is added in ice bath,followed by water (7.5 mL) dropwise. The tetrahydrofuran solution isstirred for 30 minutes, and the mixture is concentrated and diluted withwater. The white precipitate is collected, washed with water andlyophilized to give compound 5 as a white solid.

Example 5. The Preparation of Compound 6

Compound 5 (0.646 g) is mixed with TsCl (1.16 g), Et₃N (1.7 mL) andDABCO (0.23 g) in tetrahydrofuran (24 mL) and dichloromethane (50 mL).The mixture is stirred at room temperature for 24 hours andconcentrated. The residue is dissolved in dichloromethane, washed withsaturated sodium bicarbonate solution and brine, dried with sodiumsulfate, filtered and concentrated. The residue is purified by flashchromatography (hexane-dichloromethane, 50-100%) to give Compound 6 as awhite solid.

Example 6. The Preparation of Compound 7

PEG₁₁-OH (1.638 g) is added NaH (0.127 g) in tetrahydrofuran (12 mL) inice bath, and the reaction mixture is stirred for 15 minutes. To thetetrahydrofuran solution is added Compound 6 (0.666 g) intetrahydrofuran (12 mL), and the reaction mixture is stirred at roomtemperature for 24 hours. The mixture is concentrated, and mixed withwater. The suspension is extracted with EtOAc for four times, dried withsodium sulfate, filtered and concentrated. The residue is purified byflash chromatography (dichloromethane-MeOH, 0-10%) to give compound 7 aspale yellow oil.

Example 7. The Preparation of Compound 8

To the solution of Compound 7 (0.8 g) in DMF (5 mL) is addedbis(pinacolato)diboron (0.338 g), KOAc (0.26 g), followed withPd(dppf)Cl₂ (0.025 g). The reaction mixture is bubbled with Argon for 10minutes and then heated at 80° C. for 2 hours. The mixture isconcentrated, and mixed with water. The suspension is extracted withEtOAc for four times, dried with sodium sulfate, filtered andconcentrated. The residue is purified by flash chromatography(dichloromethane-MeOH, 0-10%) to give Compound 8 as pale yellow oil.

Example 8. The Preparation of Compound 10

Under the argon, to the solution of Compound 7 (0.4 g), Compound 8 (0.58g) and Compound 9 (0.021 g) in DMF (6 mL) in a Schlenk flask, K2CO3 inwater (2 M, 4 mL) is added, followed with palladiumtetrakis(triphenylphosphine) (0.018 g). The mixture is degassed viathree freeze-pump-thaw cycles, and heated to 80° C. for 24 hours. Atroom temperature, to the reaction mixture, phenylboronic acid pinacolester (0.053 g) is added under the argon, and heated to 80° C. for 2hours. At room temperature, to the reaction mixture, EDTA (0.1 g) in 20%EtOH/H2O (20 mL) is added and stirred at room temperature for 2 hours.The resulting mixture is filtered through a 0.45 μm cup filter. Thefiltered solution is diluted to the concentration of 2 mg/mL using 20%EtOH/H2O. The resulting dilution is dialyzed into 20% EtOH/H2O using atangential flow filtration system with 30 kD and 750 kD molecular weightcutoff membrane until there is less than 0.1 mg/mL of polymer in theelutant. The solution is concentrated and lyophilized to give Compound10 as a yellow solid.

Example 9. The Preparation of Compound 11 (CPCP11)

At room temperature, to the solution of Compound 10 (500 mg) indichloromethane (8 ml), trifluoroacetic acid (4 mL) is added, followedby anisole (0.05 mL). The reaction mixture is stirred at roomtemperature for 2-3 hours. The solvent is removed and dried under highvacuum overnight to give Compound 11 as pale yellow oil.

Example 10. The Preparation of Compound 22

Monomers 20 and 21 are analogously prepared using methods described bye.g., H. Feng et al. (Chemistry of Materials 2017, 29(18), 7908-7917),and polymerized with Monomer 9 as described in the procedure of Compound10 (See Example 8).

Example 11. The Preparation of Compound 23 (CPCP23)

Polymer 22 is analogously converted Polymer 23 as described in theprocedure of Compound 9 (See Example 9).

Example 12. The Preparation of CPCP Succinimidyl Ester (CPCP30)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of di(N-succinimidyl) glutarate (1 mg, AAT Bioquest) and 10μl triethylamine. The reaction mixture is stirred at room temperaturefor 2 hours, and concentrated under high vacuum to remove DMF. Theresidue is washed with ether for multiple times until most of theunreacted di(N-succinimidyl) glutarate is removed. The residue isquickly dissolved in cold acidic water (pH=5), and extracted with etherfor three times. The aqueous solution is frozen and dried to give thedesired CPCP succinimidyl ester.

Example 13. The Preparation of CPCP Maleimide (CPCP31)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of 3-maleimidopropionic acid N-hydroxysuccinimide ester (1mg, AAT Bioquest) and 10 μl triethylamine. The reaction mixture isstirred at room temperature for 2 hours, and concentrated under highvacuum to remove DMF. The residue is dissolved in acidic water (pH=5),and extracted with ethyl acetate for three times. The aqueous solutionis frozen and dried to give the desired CPCP maleimide.

Example 14. The Preparation of CPCP Dichlorotriazine (CPCP32)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of cyanuric chloride (1 mg, Sigma) and 10 μl triethylamine.The reaction mixture is stirred at room temperature for 3 hours, andconcentrated under high vacuum to remove DMF. The residue is washed withethyl acetate for multiple times until most of the unreacted cyanuricchloride is removed. The residue is quickly dissolved in cold water(pH=6), and extracted with ethyl acetate for three times. The aqueoussolution is frozen and dried to give the desired CPCP dichlorotriazine.

Example 15. The Preparation of CPCP-DBCO (CPCP33)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of DBCO-PEG4 succinimidyl ester (1 mg, AAT Bioquest) and 10μl triethylamine. The reaction mixture is stirred at room temperaturefor 3 hours, and concentrated under high vacuum to remove DMF. Theresidue is washed with ethyl acetate for multiple times until most ofthe unreacted DBCO-PEG4 succinimidyl ester is removed. The residue isdissolved in water, and extracted with ethyl acetate for three times.The aqueous solution is frozen and dried to give the desired CPCP DBCO.

Example 16. The Preparation of CPCP TCO (CPCP34)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of TCO-PEG4 succinimidyl ester (1 mg, AAT Bioquest) and 10μl triethylamine. The reaction mixture is stirred at room temperaturefor 3 hours, and concentrated under high vacuum to remove DMF. Theresidue is washed with ethyl acetate for multiple times until most ofthe unreacted TCO-PEG4 succinimidyl ester is removed. The residue isdissolved in water, and extracted with ethyl acetate for three times.The aqueous solution is frozen and dried to give the desired CPCP TCO.

Example 17. The Preparation of CPCP Methyltetrazine (CPCP35)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of methyltetrazine-PEG4 succinimidyl ester (1 mg, AATBioquest) and 10 μl triethylamine. The reaction mixture is stirred atroom temperature for 3 hours, and concentrated under high vacuum toremove DMF. The residue is washed with ethyl acetate for multiple timesuntil most of the unreacted methyltetrazine-PEG4 succinimidyl ester isremoved. The residue is dissolved in water, and extracted with ethylacetate for three times. The aqueous solution is frozen and dried togive the desired CPCP methyltetrazine.

Example 18. The Preparation of CPCP Azide (CPCP36)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of azido-PEG4 succinimidyl ester (1 mg, AAT Bioquest) and10 μl triethylamine. The reaction mixture is stirred at room temperaturefor 3 hours, and concentrated under high vacuum to remove DMF. Theresidue is washed with ethyl acetate for multiple times until most ofthe unreacted azido-PEG4 succinimidyl ester is removed. The residue isdissolved in water, and extracted with ethyl acetate for three times.The aqueous solution is frozen and dried to give the desired CPCP azide.

Example 19. The Preparation of CPCP Alkyne (CPCP37)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of alkynyl-PEG4 succinimidyl ester (1 mg, AAT Bioquest) and10 μl triethylamine. The reaction mixture is stirred at room temperaturefor 3 hours, and concentrated under high vacuum to remove DMF. Theresidue is washed with ethyl acetate for multiple times until most ofthe unreacted alkynyl-PEG4 succinimidyl ester is removed. The residue isdissolved in water, and extracted with ethyl acetate for three times.The aqueous solution is frozen and dried to give the desired CPCPalkyne.

CPCP32 can be analogously converted to thiophene-containing CPCPsuccinimidyl ester (CPCP38), CPCP maleimide (CPCP39), CPCPdichlorotriazine (CPCP40), CPCP-DBCO conjugate (CPCP41), CPCP-TCOconjugate (CPCP42), CPCP azide (CPCP43) and CPCP alkyne (CPCP44)respectively according to the procedures of Examples 12 to 19.

Example 20. The Preparation of iFluor 594-Labeled and TCO-FunctionalizedCPCP (CPCP50)

To the solution of Compound 11 (100 mg) in DMF (10 ml) is added 0.1 mlDMF solution of 4:1 iFluor 594 succinimidyl ester and iFluor 594TCO-PEG4 succinimidyl ester (1 mg, AAT Bioquest) and 10 μltriethylamine. The reaction mixture is stirred at room temperature for 3hours, and concentrated under high vacuum to remove DMF. The residue isdissolved in water, and dialyzed to remove the unreacted iFluor dyes.The aqueous solution is frozen and dried to give the desired iFluor594-labeled and TCO-functionalized CPCP. Other fluorescent dye-labeledand functionalized CCP polymers can be analogously prepared aspreviously reported (See U.S. Pat. No. 9,896,538 to Diwu et al; U.S.Pat. Nos. 8,455,613; 8,354,239; 8,362,193; and 8,575,303 to Gaylord, etal.; also WO 2013/101902 to Chiu et al).

The above examples of some synthetic strategies for the selectedpolymers of the invention, as well as their characterization, syntheticprecursors, conjugates and methods of use are provided in the examplesfor illustration. Their further modifications and permutations areobvious to one skilled in the art. For example, the second fluorophoresconjugated to the polymers of the invention can be readily selected froma large number of the commercial dyes as listed in Table 2 to make thepolymers have the different desired spectral properties. In addition,the polymers of the invention can be further functionalized with adifferent reactive functional group pairs as listed in Table 3. Thewell-known clickable groups can also be added to the polymers of theinvention for the biorthogonal chemistry-based conjugations (see P.Agarwal and R. Bertozzi, Bioconjugate Chem., 2015, 26, 176-192; K. Langand J. Chin, Chem. Reviews, 2014, 114, 4764-4806; M. D. Best,Biochemistry, 2009, 48, 6571-6584). Some other alternative methods forpolymer functionalization are well described in the literature (see U.S.Pat. Nos. 8,158,444; 8,455,613; 8,354,239; 8,362,193; and 8,575,303 toGaylord, et al.; also WO 2013/101902 to Chiu et al).

Example 21. Preparation of CPCP-Labeled Goat Anti-Mouse IgG Conjugate(CPCP51)

Goat Anti-Mouse IgG (GAM) is dissolved in 10 mM NaHCO₃ (pH 8.2) bufferto make a 5 mg/mL solution. To the aqueous GAM protein solution is addedthe DMF solution of CPCP30 (20 equivalents). The solution is rotated atroom temperature for 3 hours and the reaction mixture is transferred toan Amicon Ultra filter (MWCO=10 kDa) to remove DMF. The protein isrecovered into the initial volume with PBS buffer.

Cation exchange chromatography is used to remove free polymer.Conjugation mixture is loaded to UNOsphere™ S resin (Bio-Rad) in lowsalt buffer [50 mM MES Buffer (pH=5.0)], and incubated at roomtemperature for 10 minutes, repeatedly loading the sample for 3 times toget the maximum binding. After loading, the medium is washed with lowsalt buffer to the baseline (until the absorbance at 414 nm is lowerthan 0.01) to remove all free polymer. The retained conjugated polymerdye-GAM conjugate on the cation exchange resin is released by elevatingboth the pH and ionic strength with high salt phosphate buffer [10 mMphosphate buffer (pH=7.4)+1.0M NaCl buffer/methanol, 90/10]. Protein Aand Protein G affinity resins can also be used to remove free polymerwith comparable results. A HiTrap Protein G HP 1 mL column (GELifesciences) is pre-equilibrated with 10 mM Phosphate buffer, pH 7.4,and the SEC-purified product is slowly injected at <1 mg/mL and allowedto incubate for 30 minutes to bind. The column is washed with >10 columnvolumes of 10 mM Phosphate buffer to wash unbound polymer material offwhile monitoring absorption of the eluate at 280 nm and 414 nm to ensureall excess material is removed. The conjugate is eluted by washing thecolumn with 4 column volumes of 0.1 M Glycine pH 2.3. The elutedfractions are combined and pH-adjusted back to neutral using 1 M Tris pH8. After free polymer is removed, the conjugate solution is concentratedwith Amicon Ultra Filter (MWCO=30 kD) and loaded to a size exclusioncolumn (Superdex 200, GE life sciences) to separate conjugate andunconjugated antibody. The column is equilibrated with PBS buffer, andthe conjugated polymer-antibody conjugate is eluted before freeantibodies. For effective labeling, the degree of substitution shouldfall between 1-3 moles of conjugated polymer dye to one mole of antibodyfor most antibodies. As is well known in the art, the optima DOS dependson the properties of antibody to be labeled. The optimal labeling DOS isdetermined empirically by preparing a series of dye-conjugates over arange of DOS and comparing the desired signal/background. In some cases,a higher DOS may provide bright signal while in other cases higher DOScould reduce the affinity of the antibody to be labeled. OtherCPCP-labeled antibody conjugates (e.g., CPCP30-labeled CD45 conjugate,CPCP55) can be analogously prepared.

Example 22. CPCP-Labeled Anti-CD Antibody Conjugates for Use in FlowCytometry

Analyte-specific antibodies conjugated to a conjugated polymer dye ofthe present invention (i.e, labeled antibodies) are useful for theanalysis of blood cells (for example, in whole blood samples) by flowcytometry. The labeled-antibodies are prepared as previously described(e.g., U.S. Pat. Nos. 9,719,998; 9,758,625; 8,158,444; 8,455,613;8,354,239; 8,362,193; and 8,575,303 to Gaylord, et al.; U.S. Pat. No.9,896,538 to Diwu et al.; WO 2013/101902 to Chiu et al.). TheseCPCP-labeled antibodies can be used to stain cellular proteins, and thelabeled cells are detected using a flow cytometer. CPCP bioconjugatesare evaluated by Stain Index, as defined by BD Biosciences on a flowcytometer. See, e.g., H. Maeker and J. Trotter, BD BiosciencesApplication Note: “Selecting Reagents for Multicolour Flow Cytometry”,September 2009. The stain index reports a measure of the polymer'sbrightness, nonspecific binding. Flow cytometry provides a methodthrough which to measure cells of a specific phenotype or analytes ofinterest on specific microspheres. This can be done with direct labelingof a primary antibody or, if signal amplification is desired, through asecondary antibody or the avidin-biotin complexation with avidin-polymerconjugates. Cells of interest are taken up in sufficient quantity, spundown, washed in DPBS+0.2% BSA and 0.05% NaN₃, then resuspended instaining buffer of conjugated polymer conjugates.

Example 22. Preparation of CPCP-Labeled Phalloidin Conjugate (CPCP60)

To aminophalloidin (1 mg, AAT Bioquest) and CPCP 30 (10 mg) in DMF (0.5mL) is added N,N-diisopropylethylamine (25 μL). The mixture is stirredat room temperature for 3 hours. To this solution is added 5 mL ofEtOAc. The solid is collected by centrifugation. The crude product ispurified on SEPHADEX LH-20, eluting with water, followed by preparativeSuperdex 200 SEC column purification to give the pure phalloidinconjugate.

Example 23. The Staining of F-Actin Filaments with CPCP-LabeledPhalloidin Conjugates

Actin is a globular, roughly 42-kDa protein found in almost alleukaryotic cells. It is also one of the most highly conserved proteins,differing by no more than 20% in species as diverse as algae and humans.CPCP30-labeled Phalloidin conjugate (CPCP60) selectively binds toF-actins. Used at nanomolar concentrations, CPCP60 can be used forlabeling, identifying and quantitating F-actins in formaldehyde-fixedand permeabilized tissue sections, cell cultures or cell-freeexperiments. Cells are fixed with formaldehyde and incubated after theaddition of DMSO stock solution of CPCP conjugate. The cells are gentlyrinsed with PBS for 2 to 3 times to remove excess phalloidin conjugate.The cells are plated, sealed and imaged with a fluorescence microscope.Other CPCP conjugates can be analogously used to stain of F-actinfilaments with different fluorescence colors.

Example 24. The Preparation of Compound 52

Compound 50 is prepared as described by B. Y. Kim et al, (SeeKR2013084952). To the solution of Compound 50 (3.19 g) and TBAB (1.00 g)in toluene (80 mL) is added t-butyl acrylate (11.4 mL) in ice bath underAr protection, followed by the addition of 50% NaOH (80 mL). The mixtureis stirred at room temperature for 2.5 hours and ice-water is added. Thereaction mixture is extracted by dichloromethane twice, dried withsodium sulfate, filtered and concentrated. The residue is purified byflash chromatography (hexane-chloroform, 0-10%) to give compound 52 as awhite solid.

Example 25. The Preparation of Compound 53

To the solution of Compound 52 (8.33 g) and anisole (9.4 mL) indichloromethane (40 mL) are added trifluoroacetic acid (20 mL). Thereaction mixture is stirred at room temperature for 5 hours and thenconcentrated, azeotroped with dichloromethane twice. The residue istriturated with ether (100 mL), filtered and dried to give compound 53as a white solid.

Example 26. The Preparation of Compound 54

Compound 53 (0.93 g) is sonicated in DMF (25 mL). To the DMF solution,bromine (0.4 mL) is added in ice bath and the reaction mixture isstirred at room temperature until complete conversion to Compound 54.The mixture is poured into a sodium sulfite solution (7.5 g in 500 mLwater) with stirring. The white precipitate is collected, washed with0.1% trifluoroacetic acid solution and lyophilized to give compound 54as a white solid.

Example 27. The Preparation of Compound 55

To the solution of Compound 54 (2.19 g) in tetrahydrofuran (80 mL) isadded Et₃N (5.82 mL), then ethyl chloroformate (2 mL) in ice bath. Thereaction mixture is stirred at room temperature for 1 hour and filtered.The filtrate is concentrated and dissolved in tetrahydrofuran (90 mL).To the tetrahydrofuran solution NaBH₄ (1.05 g) is added in ice bath,followed by the dropwise addition of water (7.5 mL). The reactionsolution is stirred for 30 minutes, and concentrated and diluted withwater. The resulted precipitate is collected, washed with water andlyophilized to give compound 55 as a white solid.

Example 28. The Preparation of Compound 56

Compound 55 (0.58 g) is mixed with TsCl (1.16 g) and Et₃N (1.7 mL) intetrahydrofuran (24 mL) and dichloromethane (50 mL). The mixture isstirred at room temperature for 24 hours and concentrated. The residueis dissolved in dichloromethane, washed with saturated sodiumbicarbonate solution and brine, dried with sodium sulfate, filtered andconcentrated. The residue is purified by flash chromatography(hexane-dichloromethane, 50-100%) to give Compound 56 as a white solid.

Example 29. The Preparation of Compound 57

PEG₁₁-OH (1.64 g) is added NaH (0.13 g) in tetrahydrofuran (12 mL) inice bath, and the reaction mixture is stirred for 15 minutes. To thetetrahydrofuran solution is added Compound 56 (0.60 g) intetrahydrofuran (12 mL), and the reaction mixture is stirred at roomtemperature for 24 hours. The mixture is concentrated, and mixed withwater. The suspension is extracted with EtOAc for four times, dried withsodium sulfate, filtered and concentrated. The residue is purified byflash chromatography (dichloromethane-MeOH, 0-10%) to give Compound 57as pale yellow oil.

Example 30. The Preparation of Compound 58

To the solution of Compound 57 (0.72 g) in DMF (5 mL) is addedbis(pinacolato)diboron (0.338 g), KOAc (0.26 g), followed withPd(dppf)Cl₂ (0.025 g). The reaction mixture is bubbled with Argon for 10minutes and then heated at 80° C. for 2 hours. The mixture isconcentrated, and mixed with water. The suspension is extracted withEtOAc for four times, dried with sodium sulfate, filtered andconcentrated. The residue is purified by flash chromatography(dichloromethane-MeOH, 0-10%) to give Compound 58 as pale yellow oil.

Example 31. The Preparation of Compound 60

Under the argon, to the solution of Compound 57 (0.39 g), Compound 58(0.57 g) and Compound 9 (0.021 g) in DMF (6 mL) in a Schlenk flask,K₂CO₃ in water (2 M, 4 mL) is added, followed with palladiumtetrakis(triphenylphosphine) (0.018 g). The mixture is degassed viathree freeze-pump-thaw cycles, and heated to 80° C. for 24 hours. Atroom temperature, to the reaction mixture, phenylboronic acid pinacolester (0.053 g) is added under the argon, and heated to 80° C. for 2hours. At room temperature, to the reaction mixture, EDTA (0.1 g) in 20%EtOH/H₂O (20 mL) is added and stirred at room temperature for 2 hours.The resulting mixture is filtered through a 0.45 μm cup filter. Thefiltered solution is diluted to the concentration of 2 mg/mL using 20%EtOH/H₂O. The resulting dilution is dialyzed into 20% EtOH/H₂O using atangential flow filtration system with 30 kD molecular weight cutoffmembrane until there is less than 0.1 mg/mL of polymer in the elutant.The solution is concentrated and lyophilized to give Compound 60 as asticky yellow solid.

Example 32. The Preparation of Compound 61 (CPCP 100)

At room temperature, to the solution of Compound 60 (500 mg) indichloromethane (8 m1), trifluoroacetic acid (4 mL) is added, followedby anisole (0.05 mL). The reaction mixture is stirred at roomtemperature for 2-3 hours. The solvent is removed and dried under highvacuum overnight to give Compound 61 as pale yellow oil.

Example 33. The Functionalization of CPCP 100

CPCP100 is converted to a variety of reactive CPCP 100 derivativesanalogously as described in the procedures of Examples 12 to 20 torespectively give CPCP 100 succinimidyl ester (CPCP 110), CPCP 100maleimide (CPCP 111), CPCP 100 dichlorotriazine (CPCP 112), CPCP100-DBCO(CPCP 113), CPCP 100 TCO (CPCP114), CPCP 100 methyltetrazine (CPCP115),CPCP 100 azide (CPCP 116), CPCP 100 alkyne (CPCP117) and iFluor594-labeled and TCO-functionalized CPCP100 (CPCP118).

Example 34. The Preparation of CPCP100 Bioconjugates from theFunctionalized Reactive CPCP100 Derivatives

CPCP 100 succinimidyl ester (CPCP 110) is analogously converted to CPCP100-labeled Goat Anti-Mouse IgG Conjugate (CPCP120), CPCP 100-Labeledanti-CD antibodies and CPCP 100-Labeled Phalloidin Conjugate (CPCP 130)as described in the procedures of Examples 21 to 23.

Example 35. The Preparation of Compound 72

The solution of 70 (1.52 g) and 71 (1.37 mg) in EtOH (60 mL) is refluxedfor 15 minutes. EtOH is removed in vacuum. After 1 h under the highvacuum, the residue is dissolved in 2-propanol (60 mL) followed byadding concentrated H₂SO₄ (0.75 mL). The mixture is refluxed for 18hours. After cooling to room temperature, the pH of solution is adjustedto 10 with 1M NaOH (50 mL) that is extracted with dichloromethane (3×80mL). The combined dichloromethane solution is washed with brine anddried over anhydrous Na₂SO₄. Dichloromethane is removed in vacuum andthe residue is purified by column (10% EtOAc in Hexanes) to giveCompound 72 as white solid.

Example 36. The Preparation of Compound 76

To the solution of 72 (1.11 g) in tetrahydrofuran (10 mL) is addedt-BuOK (0.96 g) at room temperature under argon, followed by Compound 73(1.65 g). The mixture is stirred at room temperature overnight. Water(50 mL) is added to quench reaction. Tetrahydrofuran is removed invacuum. The residue is dissolved in dichloromethane, washed with brine,dried with sodium sulfate, filtered and concentrated. The residue ispurified by column (70% EtOAc in Hexane) to give Compound 76 as whitesolid.

Example 37. The Preparation of Compound 77

To the solution of 76 (0.54 g) in MeOH (30 mL), concentrated HCl (0.5mL) is added. The mixture is refluxed for 1 hour. After removing MeOH,water (50 mL) is added. The mixture is extracted with EtOAc, washed withbrine, dried with sodium sulfate, filtered and concentrated. The residueis dissolved in dichloromethane (24 mL), then TEA (1.64 mL, 11.76 mmol)and TsCl (1121 mg, 5.88 mmol) were added at room temperature. Themixture is stirred at room temperature overnight. The mixture is washedwith brine (3×20 mL). The dichloromethane layer is dried with sodiumsulfate, filtered and concentrated. The residue is purified by column(50% EtOAc in Hexane) to give Compound 77 as white solid.

Example 38. The Preparation of Compound 79

To the solution of 78 (1.95 g) in tetrahydrofuran (20 mL) is dropwiseadded 1 M t-BuOK in tetrahydrofuran (3.78 mL) at 0° C. under the argon.After 15 minutes at 0° C., the solution of Compound 77 (0.66 g) intetrahydrofuran (10 mL) is added. The mixture is stirred at roomtemperature overnight. Water (20 mL) is added to quench reaction. Afterremoving tetrahydrofuran, the suspension is extracted with EtOAc forfour times, and the combined EtOAc solution is dried with sodiumsulfate, filtered and concentrated. The residue is purified by flashchromatography (dichloromethane-MeOH, 0-10%) to give Compound 79 ascolorless oil.

Example 39. The Preparation of Compound 80

To the solution of Compound 79 (0.5 g) in DMF (3 mL) is addedbis(pinacolato)diboron (0.085 g), KOAc (0.12 g), followed withPd(dppf)Cl₂ (0.018 g). The reaction mixture is bubbled with argon for 10minutes and then heated at 80° C. for 2 hours. The mixture isconcentrated, and mixed with water. The suspension is extracted withEtOAc for four times, and the combined EtOAc solution is dried withsodium sulfate, filtered and concentrated. The residue is purified byflash chromatography (dichloromethane-MeOH, 0-10%) to give Compound 80as pale yellow oil.

Example 40. The Preparation of Compound 82

Under the argon, to the solution of Compound 80 (0.2 g) and Compound 21(0.05 g) in DMF (3 mL) in a Schlenk flask, K₂CO₃ in water (2 M, 2 mL) isadded, followed with palladium tetrakis(triphenylphosphine) (0.01 g).The mixture is degassed via three freeze-pump-thaw cycles, and heated to80° C. for 24 hours. At room temperature, to the reaction mixture,phenylboronic acid pinacol ester (0.03 g) is added under the argon, andheated to 80° C. for 2 hours. At room temperature, to the reactionmixture, EDTA (0.1 g) in 20% EtOH/H₂O (20 mL) is added and stirred atroom temperature for 2 hours. The resulting mixture is filtered througha 0.45 μm cup filter. The filtered solution is diluted to theconcentration of 2 mg/mL using 20% EtOH/H₂O. The resulting dilution isdialyzed into 20% EtOH/H₂O using a tangential flow filtration systemwith 30 kD molecular weight cutoff membrane until there is less than 0.1mg/mL of polymer in the elutant. The solution is concentrated andlyophilized to give Compound 82 as a sticky yellow solid.

Example 41. The Preparation of Compound 83 (CPCP 140)

At room temperature, to the solution of Compound 82 (200 mg) indichloromethane (4 ml), trifluoroacetic acid (2 mL) is added, followedby anisole (0.05 mL). The reaction mixture is stirred at roomtemperature for 2-3 hours. The solvent is removed and dried under highvacuum overnight to give Compound 83 as pale yellow oil.

Example 42. The Functionalization of CPCP 140

CPCP200 is converted to a variety of reactive CPCP 200 derivativesanalogously as described in the procedures of Examples 12 to 17 torespectively give CPCP 200 succinimidyl ester (CPCP 150), CPCP 200maleimide (CPCP 151), CPCP 200 dichlorotriazine (CPCP 152), CPCP200-DBCO(CPCP 153), CPCP 200 TCO (CPCP154), CPCP 200 methyltetrazine (CPCP155),CPCP 200 azide (CPCP 156), CPCP 200 alkyne (CPCP157) and iFluor594-labeled and TCO-functionalized CPCP200 (CPCP158).

Example 43. The Preparation of CPCP200 Bioconjugates from theFunctionalized Reactive CPCP200 Derivatives

CPCP 200 succinimidyl ester (CPCP 150) is analogously converted to CPCP200-labeled goat anti-mouse igg conjugate (CPCP160), CPCP 100-labeledanti-CD antibodies and CPCP 200-Labeled Phalloidin Conjugate (CPCP 170)as described in the procedures of Examples 18 to 21.

For primary incubation, cells are incubated with a primary conjugatespecific to an antigen of interest, negative cells served as a negativenon-specific binding reference. A control population or an establishedcommercial conjugate is used as a positive control. Primaryantibody-polymer conjugates are incubated at various concentrations withvolume dilutions typically from 10 nM-500 uM for 30 minutes.

For secondary antibody labeling, an unlabeled primary antibody to theantigen of interest is incubated at 1-50 μg/ml, or other titratedamount. Following primary incubation, cells are rinsed with 5 volumesstaining buffer and spun down for 3-5 minutes. Species reactivesecondary conjugated polymer conjugates are incubated at concentrationswith volume dilutions from 10-500 nM for 30-60 minutes. Followingsecondary incubation, cells are rinsed with 3-5 volumes staining bufferand spun down for 3-5 minutes. Cells are resuspended for testing inDPBS+0.2% BSA, 0.05% sodium azide.

For streptavidin-polymer conjugate labeling, cells are incubated with abiotinylated primary antibody to the marker of interest, as detailedabove for the secondary antibody labeling, instead of an unlabeledprimary. Following the primary washing, cells are resuspended andincubated with streptavidin-polymer conjugates at 1-100 nM volumedilutions for 30 minutes. Following secondary incubation, cells arerinsed with 5 volumes staining buffer and spun down for 3-5 minutes.Cells are resuspended for testing. If further signal amplification isdesired, cells could be incubated with an unlabeled primary antibody andthen subsequently followed with a species reactive biotinylatedsecondary antibody prior to incubation with streptavidin conjugates.

It will be understood that the particular antibody conjugate used andthe specific reaction components and particular reaction conditions usedcan have an effect on the results obtained. Routine experimentationshould be carried out to determine preferred reaction components, suchas buffers or lyse solutions, and reaction conditions, includingstaining times and temperatures. Such routine optimization of assayconditions is standard practice in the field of immunostaining-basedassays.

What is claimed is:
 1. A conjugated polymer of Formula 1 comprising aconjugated segment composed of x distinct comonomer units (Mr-1 to Mr-x)that are randomly distributed along the conjugated polymer backbone:

wherein: Mr-1 is a comonomer of Formula 11a:

wherein: R₃ to R₅ are independently selected from hydrogen, a halogen,an alkyl, a water solubilizing group (WSG), an aryl, a heteroaryl group,a fluorescent dye (FD), a functional group (FG), and linked biologicalsubstrate (L-BS); Y and Z are independently selected from O, S, N—R₁₃,P—R₁₃, O═P—R₁₃, O═P—OR₁₃, O═S—R₁₃, and O═S(O)—R₁₃; R₁₁-R₁₆ areindependently selected from hydrogen, an alkyl, a substituted alkyl, aWSG, linked WSG (L-WSG), an aryl, a heteroaryl group, a FD, a FG, alinked FG (L-FG), and L-BS; the Mr-1 comonomer units are connected toadjacent comonomers of the conjugated polymer backbone through positionsC1 and C7 of the Mr-1 comonomer; and at least two of R₁₁ to R₁₆ is a WSGor comprises a WSG; Mr-2 to Mr-x are distinct comonomer units eachindependently selected from ethenylene, acetylene, an aryl, aheteroaryl, and a fluorescent dye FD (FD); m1 is an integer greater thanor equal to 5; and m2 to mx are each independently an integer from 0 to200; wherein: (1) the sum of ml through mx is >10; and (2) optionally,at least one comonomer of Mr to Mr-X comprises a functional group (FG)or linked biological substrate (L-BS).
 2. The conjugated polymer ofclaim 1, wherein at least one of the Mr-1 to Mr-x co-monomers is afluorescent dye (FD) or comprises a linked FD configured in energyreceiving proximity to the conjugated polymer.
 3. The conjugated polymerof claim 1, wherein the terminal comonomers of the conjugated polymerbackbone are optionally and independently capped with phenyl, aryl,heteroaryl or a substituted version thereof that is substituted withbromo, iodo, boronyl, a FG or L-BS.
 4. The conjugated polymer of claim1, wherein the linker (L) comprises one or more groups selected from analkyl, a PEG, a carboxamide, a thioether, an ester, an imine, ahydrazine, an oxime, an alkyl amine, an ether, an aryl amine, a boronateester, an N-acylurea or anhydride, a platinum complex, an aminotriazine,a triazinyl ether, an amidine, a urea, a urethane, a thiourea, aphosphite ester, a silyl ether, a sulfonamide, a sulfonate ester, a1,2,3-triazole, a pyradazine, a thiazolidine, a2-diphenylphosphonyl-benzoamide, an isoxazole and a succinimide group.5. The conjugated polymer of claim 1, wherein R₃ to R₅ are independentlyselected from hydrogen, fluoro, chloro, and alkyl.
 6. The conjugatedpolymer of claim 1, wherein one or more of the WSG comprises a PEG groupselected from PEG6 to PEG18.
 7. The conjugated polymer of claim 1,wherein at least one of the Mr-1 to Mr-x co-monomers is a fluorescentdye (FD) or comprises a linked FD, wherein the FD is a fluorescein, arhodamine, a rhodol, a cyanine, a bodipy, a squaraine, a coumarin, aperylenediimide, a diketopyrrolopyrrole, a porphyrin or aphthalocyanine.
 8. The conjugated polymer of claim 5, wherein R₃ to R₅are each hydrogen.
 9. A method of detecting an analyte in a sample,comprising a) contacting said sample with a detection reagent comprisinga conjugated polymer according to claim 1 under conditions in which saiddetection reagent binds said analyte, if present, to produce a detectionreagent-bound analyte; and b) detecting whether the detectionreagent-bound analyte is present using fluorescence of the conjugatedpolymer.
 10. The method of claim 9, wherein: the detection reagent is anantibody; the detection reagent is an anti-digoxigenin antibody; thedetection reagent is a goat anti-mouse IgG antibody, goat anti-rabbitIgG antibody, goat anti-human IgG antibody, donkey anti-mouse IgGantibody, donkey anti-rabbit IgG antibody, donkey anti-human IgGantibody, chicken anti-mouse IgG antibody, chicken anti-rabbit IgGantibody, or chicken anti-human IgG antibody; or the detection reagentis an avidin, streptavidin, neutravidin, avidinDN, or avidinD moiety.11. The method according to claim 9, wherein the analyte is a targetprotein expressed on a cell surface.
 12. The conjugated polymer of claim4, wherein the linker comprises an alkyl or a PEG group.
 13. Theconjugated polymer of claim 1, wherein at least one comonomer of Mr toMr-X comprises a linked biological substrate (L-BS), wherein: L is analkyl chain or a PEG chain; and BS is an antibody, a peptide, a protein,an oligonucleotide, a nucleic acid or a carbohydrate.
 14. The conjugatedpolymer of claim 13, wherein BS is an antibody.
 15. The conjugatedpolymer of claim 1, wherein R¹¹ to R¹⁶ are independently selected from aPEG, an alkyl, a carboxyalkyl, a sulfonylalkyl, a phosphonylalkyl, anaminoalkyl and L-BS.
 16. The conjugated polymer of claim 1, wherein R¹¹to R¹⁶ are independently selected from H, alkyl, substituted alkyl, WSG,and linked WSG (L-WSG), wherein: L is an alkyl chain; and WSG is a PEGgroup.
 17. The conjugated polymer of claim 1, wherein Y and Z are eachindependently selected from O, S, and N—R₁₃.
 18. The conjugated polymerof claim 17, wherein Y and Z are each S.
 19. The conjugated polymer ofclaim 17, wherein Y and Z are each O.